JP2015232022A - Newcastle disease virus vectored avian vaccines - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide Newcastle disease virus (NDV) vaccines or compositions which can protect animals (particularly avian) from diseases.SOLUTION: The invention includes engineered Newcastle disease virus (NDV) vaccines or compositions. The invention also includes recombinant vectors encoding and expressing avian pathogenic antigens, more specifically avian influenza proteins, epitopes or immunogens. The invention provides compositions in which the above mentioned engineered NDV vector comprises a polynucleotide having at least 90% sequence identity to a polynucleotide having the sequence as set forth in SEQ ID NO:1 or a polynucleotide complementary to a polynucleotide having at least 90% sequence identity to the polynucleotide having the sequence as set forth in SEQ ID NO:1.

Description

Inclusion this application to herein by reference claims the rights of US Provisional Patent Application No. 61 / 166,481 (April 03, 2009 application).
The invention encompasses NDV vector avian vaccines or compositions, specifically avian influenza vaccines. The vaccine can be an engineered avian vaccine.

Several recent studies have highlighted the potential of Newcastle disease virus (NDV) that can be used as a vaccine vector for avian diseases (Krishnamurthy et al., Virology 278, 168-182,2000; Huang et al., J. Gen. Virol. 82, 1729-1736, 2001; Nakaya et al., J. Virol. 75, 11868-11873, 2001; Park et al. PNAS 103, 8203-8208, 2006; Veits et al PNAS 103, 8197-8202, 2006; Ge et al. J. Virol. 81, 150-158, 2007; Romer-Oberdorfer et al. Vaccine 26, 2307-2313, 2008).
NDV belongs to the family Paramyxoviridae and Avravirus. NDV replicates in the respiratory tract, gastrointestinal tract, fallopian tube, and for some isolates in the nervous system. Transmission is aerogenic and by the oral and fecal routes. NDV causes highly contagious lethal diseases that affect all avian species and can also infect several mammalian species. Depending on the virus strain and host species, the disease can vary from clinically unclear to highly toxic forms. NDV strains can be classified into three different pathological types according to the continuous spectrum of virulence of NDV strains: lentogenic, mesogenic and velogenic (Alexander, DJ, Diseases of Poultry, Iowa State Uni. Press , Ames IA, 541-569, 1997). Roentogenic strains do not normally cause disease in adult chickens and are widely used as live vaccines in the poultry industry in the United States and other countries. Viruses with intermediate virulence are termed mesogenic, while viruses that cause high mortality are termed velogenic. The disease is distributed throughout the world and continues to be a major and persistent threat to industrial poultry production.

The NDV genome is an approximately 15 kb non-segmented minus-strand RNA. This genomic RNA contains six genes that encode the following proteins in the following order: nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin − Neuraminidase (HN) and large polymerase protein (L). Two other proteins V and W with unknown function are generated by RNA editing during P gene transcription (Steward et al., 1993, Journal of General Virology 74: 2539-2547).
The development of a recently established method for the complete recovery of non-segmented negative RNA viruses from cloned cDNA opens the possibility of genetically manipulating this virus group (including NDV) (Conzelmann, KK Rev. Genet. 32, 123-162, 1998; Roberts and Rose, Virology 247, 1-6, 1998). This unique molecular genetic methodology (called “reverse genetics”) not only elucidates the function of various viral coding genes (Palese et al., PNAS 93, 11354-11358, 1996; Nagai , Y., Rev. Med. Virol. 9, 83-99, 1999) also provide a means to allow these viruses to be used to express heterologous genes (Bukreyev et al., J. Virol 70, 6634-6641, 1996; Mebatsion et al., PNAS 93, 7310-7314, 1996; Schnell et al., PNAS 93, 11359-11365, 1996; Hasan et al., J. Gen. Virol. 78, 2813-2820, 1997; He et al., Virology 237, 249-260, 1997; Sakai et al., FEBS Lett. 45, 221-226, 1999). The foregoing provides methods for making improved vaccines and vaccine vectors.

  A recovery system derived from cloned cDNA, based on the NDV lentogenic vaccine strain (LaSota), was simultaneously reported in 1999 by two separate groups (Peeters et al., 1999; Romer-Oberdorfer et al ., 1999). In the first reported system, the full-length NDV cDNA from LaSota strain (ATCC-VR699) was assembled with a pOLTV5 transcription vector containing a T7 DNA-dependent RNA polymerase promoter. Individual NDV transcriptase complex (NP, P and L) clones were cloned in eukaryotic expression vectors. The cotransfection protocol generated several infectious centers in infected monolayer cultures (Peeters et al., J. Virol. 73, 5001-5009, 1999). A second system reported to recover roentogenic NDV from cloned cDNA assembles a full-length antigenome expression plasmid and support plasmid (Romer-Oberdorfer et al., Journal of General Virology, 80, 2987-2995, 1999) using essentially the same strategy. Other systems have recently been developed to recover the lentogenic NDV Hitchner B1 (Nakaya et al. 2001) or LaSota strain (Huang et al. 2001). The only system available for the recovery of recombinant mesogenic NDV was described by Kurishnamurthy (2000). This system utilized vaccinia virus recombinant (MVA) and HEP-2 cells for transfection. Full length clones of the mesogenic strain Beaudette C and support plasmids (N, P and L) from the same strain were used for transfection. An additional transcription unit encoding the CAT reporter gene was placed between the HN and L genes. The growth of rNDV expressing the CAT gene was slow and the virus was attenuated. The CAT gene was stably expressed for several passages in cell culture.

  Avian influenza (AIV) (sometimes called Avianflu, commonly recognized as Triinflu) refers to influenza caused by an influenza virus that has acclimatized to birds. AIV is a segmented single-stranded negative-sense RNA virus belonging to the Orthomyxoviridae family, and is classified as influenza A virus. Type A virus is the most common cause of animal and human influenza. This type occurs in a number of strains or subtypes, which are distinguished on the basis of two surface lipid envelope membrane proteins, hemagglutinin (HA) and neuraminidase (NA). HA facilitates virus entry into the host cell and NA helps release the progeny virus of the infected cell (de Jong et al., J Clin Virol. 35 (1): 2-13, 2006). Influenza A viruses are classified into subtypes based on their unique HA and NA content. There are 16 different HA subtypes and 9 different NA subtypes. Many different combinations of HA and NA proteins are possible. Influenza A virus subtypes are named according to their HA and NA surface proteins. For example, “H7N2 virus” refers to an influenza A subtype having an HA protein of the H7 subtype and an NA protein of the N2 subtype. Similarly, the “H5N1” virus has H5 subtype HA and N1 subtype NA. The H5N1 subtype is clearly associated with recent epidemics in Asia, Russia, the Middle East, Europe and Africa (Olsen et al., Science 21; 312 (5772): 384-8, 2006).

Influenza A virus can infect humans, pigs, horses, seals, whales, poultry, cats, dogs, weasels and other animals, but wild birds are their natural hosts. Water birds constituted the major influenza-bearing animals from which the virus lineage evolved and adapted to their hosts (eg, human, swine and equine influenza). Host specificity is not absolute and interspecies transmission can occur as illustrated by the ability of highly pathogenic avian influenza (HPAI) H5N1 subtypes to infect human, cat, dog and pig species.
The highly pathogenic influenza A virus subtype H5N1 virus is a newly emerging avian influenza virus that is of global concern as a potential threat of an outbreak. H5N1 kills millions of poultry in many countries in Asia, Europe and Africa where its number continues to grow. Unlike influenza B, influenza A causes an antigenic shift (at least two virus strains together form a new subtype), epidemiologists, infectious disease researchers, and other health-related experts Is a novel virulent strain of influenza that is easily transmitted and lethal to humans because the coexistence of human influenza virus and avian influenza (especially H5N1) provides an opportunity for the exchange of genetic material between species-specific viruses (Food Safety Research Information Office, “A Focus on Avian Influenza”. May 2006, updated November 2007).
Since the first outbreak of H5N1 occurred in 1997, the number of bird-to-human transmissions of HPAI H5N1 resulting in clinically significant and fatal human infections has increased. However, viruses do not easily reach humans because of the large species barrier that exists between birds and humans. Millions of birds have been infected with the virus since its discovery, but only about 200 people in Indonesia, Laos, Vietnam, Romania, China, Turkey and Russia have died from avian influenza.
Given the susceptibility of animals (including humans) to AIV, a means to prevent AIV and protect animals is essential. Therefore, there is a need for an effective vaccine against influenza.
Citation or certification of any record in this application is not an admission that such record is available as prior art to the present invention.

  The present invention provides an effective avian vaccine or composition, a method of effectively immunizing birds.

The present invention is based in part on Applicants' discovery that the AIV hemagglutinin gene expressed by the enterotrophic AVINEW ™ strain of Newcastle disease virus (NDV) is highly immunogenic in birds. Yes.
The present invention relates to an avian vaccine or composition using NDV as a vector, which contains a specific avian antigen (more specifically, avian influenza antigen) and has an inherent intestinal tropism that expresses it. An effective amount of NDV vector and a pharmaceutically or veterinarily acceptable carrier, excipient or vehicle can be included. The intestinal NDV may be an AVINEW ™ modified live vaccine NDV strain marketed by Merial Limited.
The avian influenza antigen can be hemagglutinin. The avian influenza HA antigen can be H5 subtype HA.
The present invention also relates to a method of immunizing a bird, said method comprising an effective amount of a vaccine that can comprise an effective amount of a recombinant NDV vector and a pharmaceutically or veterinarily acceptable carrier, excipient or vehicle. The step of administering. Administration can be by in ovo, instillation, spray, drinking water, or parenteral (subcutaneous, intramuscular, transdermal) administration.
The present invention provides a method of modifying an Avinew NDV genome to produce an engineered Avinew NDV, the method comprising introducing an isolated polynucleotide into the Avinew NDV genome into a non-essential region of the Avinew NDV genome. Said non-essential region is an open reading frame encoding a non-essential protein or a non-essential part of the open reading frame; or upstream of the NP gene of the Avinew NDV genome, or between two genes (intergenic region), or of the L gene It may be a non-translated (or non-coding) region located downstream.

The invention further relates to the administration of a vaccine or composition using a prime-boost protocol. The present invention primes birds with an avian influenza vaccine prior to administration of a vaccine of the present invention (which can include an effective amount of an engineered NDV vector and a pharmaceutically or veterinarily acceptable carrier, excipient or vehicle). About that. Alternatively, the invention further relates to priming with a vaccine of the invention (an engineered NDV vector that expresses at least one avian influenza antigen and a pharmaceutically or veterinarily acceptable carrier) prior to administration of the avian influenza vaccine.
The present invention further includes a kit for carrying out a method for inducing or inducing an immune response, the kit being either an immunological composition or vaccine of recombinant influenza, or an inactivated immunological composition or vaccine. And instructions for performing the method.
Accordingly, it is an object of the present invention that none of the previously known products, methods of making the products, or methods of using the products are included within the scope of the present invention, and thus applicants Reserve the right to express waiver of any previously known product, process or method rights. Furthermore, the present invention relates to any product, process, or production of said product that does not meet the description and authorization requirements of USPTO (35 USC Section 112, first paragraph) or EPO (EPC Chapter 83). Neither method nor use of the product is included within the scope of the present invention, so Applicants reserve the right to them and thereby any of the products previously described, the product Or the method of using the product is excluded from the present invention.
The foregoing and other embodiments are described in, or are apparent from, the following detailed description, and are included in the description.

FIG. 1A shows a genetic map of the full-length NDV genome. FIG. 1B shows genetic maps of two engineered NDVs with influenza HA inserted at two representative transgene insertion sites in the full-length NDV genome. FIG. 1C is an example of a flowchart of the NDV reverse genetics system. FIG. 1D shows a method for recovering engineered NDV infectious particles using NDV reverse genetics. Schematic showing the insertion of unique restriction sites (PacI and FseI) allowing cloning of the entire AVINEW genome in a transcription plasmid for reverse genetics development and easy cloning of the transgene between P and M. . The plasmid map of plasmid pIV029 is shown. 4a-4o provides the sequence of the insert of plasmid pIV029. Italic type corresponds to non-AVINEW ™ sequences, underlined italic type corresponds to internal PacI-FseI insertion between P and M genes; italic type corresponds to HDV ribozyme (3 'end), and The underlined italics correspond to the T7 promoter (5 'end) and terminator (3' end). A transcription start sequence (GS = Gene Start (gene start point)) and a transcription stop sequence (GE = Gene End (gene end point)) are shown in the map, and the sequence is underlined. The circled T (position 3190 in FIG. 4c) corresponds to the enclosed nucleotide (before PacI-FseI insertion), which is the T (SEQ ID NO :) in the AVINEW ™ genome assembled into a transcription vector. (Position 3190 of 24) (T is derived from the primer used to create the PacI-FseI restriction site). In the consensus AVINEW ™ genome (SEQ ID NO: 1), the corresponding nucleotide at position 3150 is C. 4a continued. Fig. 4b continued. FIG. 4c continued. FIG. 4d continued. FIG. 4e continued. FIG. Fig. 4g continued. FIG. 4h continued. FIG. 4i continued. FIG. Fig. 4k continued. Fig. 4l continued. 4m continued. 4n continued. The plasmid map of plasmid pIV32 is shown. The plasmid map of plasmid pIV33 is shown. The plasmid map of plasmid pIV034 is shown. The plasmid map of plasmid pNS151 is shown. A schematic method for introducing the AIV HA gene into the NDV genome by NDV insertion cassette is shown. The plasmid map of plasmid pIV039 is shown. 11A shows HA expression by engineered NDV vAVW02. VAVW01 without insert is used as a negative control. HA expression is detected by Western blot in the urinary sac of virus-inoculated embryos (left panel in FIG. 11A) or in infected CHO cell lysates (right panel in FIG. 11A). 11B shows HA expression by engineered NDV vAVW02. VAVW01 without insert is used as a negative control. HA expression is detected by immunofluorescence of infected cells using chicken serum that is anti-H5 positive in infected CHO cells (FIG. 11B). Figure 2 is a table showing SEQ ID NOs assigned to DNA and protein sequences. 1 shows the HI titers of anti-MDV and anti-AI (to either H5N1 Cl2 or H5N9LP antigen) maternal antibody (MDA) of 1 day offspring chickens of various groups of vaccine immunized SPF chickens. Figure 2 shows the time course and protocol of vaccine immunization / challenge for assessing avian influenza protection induced by engineered NDV in SPF chickens. Day-of-age vaccinated with vAVW01 (v01 or 01; no inserted gene) or vAVW03 (v03 or 03; AI HA inserted), no MDA (SPF) or NDV MDA (21A), or NDV and H5 The kinetics diagram of the survival rate of chickens with MDA or H5 only NDA (21B) is shown. A comparison of AIVs emanating from oral swabs (cotton swabs) (A and C) and total excretory swabs of chickens without (DAF) vaccinated with vAVW01 or vAVW03 (SPF) or with MDA is shown. C and D results are expressed as the log10 ratio of the average level of virus shedding by HPAI H5N1 challenge of vAVW01-immune chickens and vAVW03-immune chickens at various time points after challenge. A comparison of AIVs emanating from oral swabs (cotton swabs) (A and C) and total excretory swabs of chickens without (DAF) vaccinated with vAVW01 or vAVW03 (SPF) or with MDA is shown. C and D results are expressed as the log10 ratio of the average level of virus shedding by HPAI H5N1 challenge of vAVW01-immune chickens and vAVW03-immune chickens at various time points after challenge. A comparison of AIVs emanating from oral swabs (cotton swabs) (A and C) and total excretory swabs of chickens without (DAF) vaccinated with vAVW01 or vAVW03 (SPF) or with MDA is shown. C and D results are expressed as the log10 ratio of the average level of virus shedding by HPAI H5N1 challenge of vAVW01-immune chickens and vAVW03-immune chickens at various time points after challenge. A comparison of AIVs emanating from oral swabs (cotton swabs) (A and C) and total excretory swabs of chickens without (DAF) vaccinated with vAVW01 or vAVW03 (SPF) or with MDA is shown. C and D results are expressed as the log10 ratio of the average level of virus shedding by HPAI H5N1 challenge of vAVW01-immune chickens and vAVW03-immune chickens at various time points after challenge. Shows the effect of NDV MDA on vAVW03-induced AIV HI titers (using H5N9 (A) clade 2.2 (B) antigen) after vaccine immunization (D21) and after challenge (D35) (SPF: no MDA; NDV: anti-NDV MDA). The numbers entered in the figure correspond to the number of positive sera / total number of tests. In the presence of NDV MDA, AIV HI titers were higher after vaccine immunization compared to SPF chickens without NDV MDA, but did not increase after challenge. Figure 2 shows the effect of NDV MDA on vAVW03-induced AIV HI titers (using H5N1 clade 2.2 (B) antigen) after vaccine immunization (D21) and after challenge (D35) (SPF: no MDA; NDV: anti-NDV MDA). The numbers entered in the figure correspond to the number of positive sera / total number of tests. In the presence of NDV MDA, AIV HI titers were higher after vaccine immunization compared to SPF chickens without NDV MDA, but did not increase after challenge. NDV HI titers in D21 after vaccination of chickens with no MDA (SPF), AI H5N9 and NDV MDA (H5N9 + NDV), NDV MDA (NDV), or AI H5N1 MDA (H5N1) with vAVW01 or vAVW03 Indicates. NDV MDA had no negative effect on the level of NDV HI titers induced with vAVW01 or vAVW03. NDV HI titers after immunizing D0 and D21 twice with AVINEW (G1), vAVW02 (G2) and vAVW03 (G3) are shown. Shown are AI H5N1 HI titers after vaccination of D0 and D21 twice with vAVW02 (G2) or vAVW03 (G3). Shown are AI H5N1 SN titers after vaccination of D0 and D21 twice with vAVW02 (G2) or vAVW03 (G3). 22A shows NDV HI titers induced by one (D0) or two (D0 and D14) ophthalmic administration of vAVW03. 22B shows the AIV SN titer induced by one (D0) or two (D0 and D14) ophthalmic administration of vAVW03. 23A-23D is unvaccinated (Group 1) or vaccinated (Group 2: vAVW03 administered once to D0; Group 3: vAVW03 administered twice to D0 and D14; Group 4: vFP89 and D14 administered to D0 VAVW03D28 prime-boost), Muscovy duck chick oropharyngeal swab (A and C) and total excretory swab (B and D) viral loads (A and B) challenged with HPAI H5N1 isolate on day 28 ) And the percentage of positive samples (C and D). 23A-23D is unvaccinated (Group 1) or vaccinated (Group 2: vAVW03 administered once to D0; Group 3: vAVW03 administered twice to D0 and D14; Group 4: vFP89 and D14 administered to D0 VAVW03D28 prime-boost), Muscovy duck chick oropharyngeal swab (A and C) and total excretory swab (B and D) viral loads (A and B) challenged with HPAI H5N1 isolate on day 28 ) And the percentage of positive samples (C and D). 23A-23D is unvaccinated (Group 1) or vaccinated (Group 2: vAVW03 administered once to D0; Group 3: vAVW03 administered twice to D0 and D14; Group 4: vFP89 and D14 administered to D0 VAVW03D28 prime-boost), Muscovy duck chick oropharyngeal swab (A and C) and total excretory swab (B and D) viral loads (A and B) challenged with HPAI H5N1 isolate on day 28 ) And the percentage of positive samples (C and D). 23A-23D is unvaccinated (Group 1) or vaccinated (Group 2: vAVW03 administered once to D0; Group 3: vAVW03 administered twice to D0 and D14; Group 4: vFP89 and D14 administered to D0 VAVW03D28 prime-boost), Muscovy duck chick oropharyngeal swab (A and C) and total excretory swab (B and D) viral loads (A and B) challenged with HPAI H5N1 isolate on day 28 ) And the percentage of positive samples (C and D). 24A-24D is vaccinated (group 1) or vaccinated (group 2: vAVW03 administered once to D0; group 3: vAVW03 administered twice to D0 and D14; group 4: vFP89 and D14 administered to D0 VAVW03D28 prime-boost), Muscovy duck chick oropharyngeal swab (A and C) and total excretory swab (B and D) viral loads (A and B) challenged with HPAI H5N1 isolate on day 42 ) And the percentage of positive samples (C and D). 24A-24D is vaccinated (group 1) or vaccinated (group 2: vAVW03 administered once to D0; group 3: vAVW03 administered twice to D0 and D14; group 4: vFP89 and D14 administered to D0 VAVW03D28 prime-boost), Muscovy duck chick oropharyngeal swab (A and C) and total excretory swab (B and D) viral loads (A and B) challenged with HPAI H5N1 isolate on day 42 ) And the percentage of positive samples (C and D). 24A-24D is vaccinated (group 1) or vaccinated (group 2: vAVW03 administered once to D0; group 3: vAVW03 administered twice to D0 and D14; group 4: vFP89 and D14 administered to D0 VAVW03D28 prime-boost), Muscovy duck chick oropharyngeal swab (A and C) and total excretory swab (B and D) viral loads (A and B) challenged with HPAI H5N1 isolate on day 42 ) And the percentage of positive samples (C and D). 24A-24D is vaccinated (group 1) or vaccinated (group 2: vAVW03 administered once to D0; group 3: vAVW03 administered twice to D0 and D14; group 4: vFP89 and D14 administered to D0 VAVW03D28 prime-boost), Muscovy duck chick oropharyngeal swab (A and C) and total excretory swab (B and D) viral loads (A and B) challenged with HPAI H5N1 isolate on day 42 ) And the percentage of positive samples (C and D). Provides sequence alignment between avian influenza HA proteins and amino acid level sequence identity percentage. Fig. 25-1 continued. Fig. 25-2 continued. Provides sequence alignment between avian influenza HA proteins and nucleic acid level sequence identity percentage. Fig. 26-1 continued. Fig. 26-2 continued. Fig. 26-3 continued. Fig. 26-4 continued. Fig. 26-5 continued.

The following detailed description, which is provided by way of example but is not intended to limit the invention only to the specific embodiments described, is best understood in conjunction with the accompanying drawings. Like.
In the present disclosure, the following should be noted, particularly in the claims. For example, the term “includes” (“comprises”, “comprised”, “comprising”, etc.) has the meaning ascribed to them in US patent law, for example, the terms “includes”, “ included "," including ", etc.). In addition, terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in US patent law, for example, Ingredients not listed are allowed, but ingredients found in the prior art or affecting basic or novel features of the invention are excluded.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms “a”, “an”, and “the” include the plural counterparts unless the context clearly indicates otherwise. Similarly, the term “or” is intended to include “and” unless the context clearly indicates otherwise.

In the present invention, the AVINEW vaccine strain is used as a vector. AVINEW is a live NDV vaccine (Merial Limited) widely used throughout the world. This vaccine strain is naturally non-pathological and exhibits a respiratory and intestinal duality. Furthermore, the AVINEW strain belongs to the NDV gene group (class II, genotype I) that can infect ducks. In contrast to LaSota, whose tropism is essentially airways, AVINEW strains do not induce respiratory side reactions.
The present invention relates to an avian vaccine that can comprise an effective amount of an engineered NDV vector and a pharmaceutically or veterinarily acceptable carrier, excipient or vehicle.
The invention encompasses any engineered NDV vector that expresses a protein, polypeptide, antigen, epitope or immunogen that elicits an immunological response in an animal (eg, a bird). Said protein, polypeptide, antigen, epitope or immunogen is an influenza protein, polypeptide, antigen, epitope or immunogen, eg, a protein, polypeptide, peptide or that elicits, induces or stimulates a response in an animal (eg a bird) It can be (but is not limited to) a fragment thereof.
“Animal” is intended to include mammals and birds. Animals or hosts include mammals and humans. The animals may be equine (eg, horse), canine (eg, dog, wolf, fox, coyote, jackal), feline (eg, lion, tiger, domestic cat, wild cat, other large cats) And other felines (including cheetahs and lynx)), sheep (eg sheep), bovines (eg cattle), pigs (eg pigs), birds (eg chickens, ducks, cancers) , Turkey, quail, pheasant, parrot, finch, hawk, crow, ostrich, emu, and cassowary), primate (eg, primate, tarsier, tailed monkey, gibbon, anemone), and fish You can choose from. The term “animal” includes individual animals of all developmental stages, including embryonic and fetal stages.

In certain embodiments, the avian influenza immunological composition or vaccine comprises an engineered vector and a pharmaceutically or veterinarily acceptable carrier, excipient or vehicle. The engineered vector can be an NDV expression vector that can include a polynucleotide encoding an influenza protein, polypeptide, antigen, epitope or immunogen. The influenza protein, polypeptide, antigen, epitope or immunogen may be hemagglutinin, matrix protein, neuraminidase, nonstructural protein, nucleoprotein, polymerase, or any fragment described above.
In another embodiment, the influenza protein, polypeptide, antigen, epitope or immunogen may be derived from an avian or avian influenza strain infected with influenza. Avian influenza proteins, antigens, epitopes or immunogens include hemagglutinin (HA) (e.g. HA precursor, H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16 protein (but not limited to)), matrix protein (eg, matrix protein M1 or M2 (but not limited to)), neuraminidase (eg, NA1, NA2, NA3, NA4, NA5, NA6, NA7, NA8 or NA9 (but not limited to), non-structural (NS) protein (such as but not limited to NS1 or NS2), nucleoprotein (NP) and polymerase (such as PA polymerase, PB1 polymerase 1 or PB2) Polymerase 2 (but not limited to)).

The avian influenza antigen can be a hemagglutinin, such as H5 HA. In one embodiment, H5 is H5N1 A / turkey / Turkey 1/2005 (Clade 2.2), A / chicken / Indonesia / 7/2003 (Clade 2.1), A / Duck / Laos / 3295/2006 (Clade 2.3), A Isolated from the strain / Chicken / West Java / PWT-WIJ / 2006 (Clade 2.1).
In another embodiment, the avian influenza protein, polypeptide, antigen, epitope or immunogen may be derived from an avian influenza strain derived from an influenza-infected bird or a recent isolate of any subtype.
As used herein, “antigen” or “immunogen” refers to a substance that induces a specific immune response in a host animal. An antigen is an entire organism (killed, attenuated or live); a subunit or part of an organism; a recombinant comprising an insert that expresses an epitope, polypeptide, peptide, protein or fragment thereof having immunogenic properties. A vector; a fragment or fragment of a nucleic acid capable of inducing an immune response when provided to a host animal; a protein, polypeptide, peptide, epitope, hapten, or any combination of the foregoing. Alternatively, the immunogen or antigen can include a toxin or antitoxin.
As used herein, the term “immunogenic protein or peptide” also refers to that once administered to a host, it can cause an induced humoral and / or cellular immune response type. Peptides and polypeptides that are immunologically active in the sense are included. Preferably, the protein fragment is one that has substantially the same immunological activity as the total protein. Accordingly, a protein fragment of the invention comprises, consists essentially of, or consists of at least one epitope or antigenic determinant. The term epitope (also known as an antigenic determinant) is the part of a macromolecule that is recognized by the immune system and is a humoral (B cell) and / or cellular (T cell) immune response Can be induced.

  The term “immunogenic protein or peptide” also contemplates deletions, additions and substitutions of the sequence so long as the polypeptide functions to produce an immunological response as defined herein. In this regard, particularly preferred substitutions will generally be substitutions that are conservative in nature, ie occur within the amino acid family. For example, amino acids are generally divided into four families: (1) acidic—aspartic acid and glutamic acid; (2) basic—lysine, arginine, histidine; (3) nonpolar—alanine, valine, leucine, Isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar-glycine, asparagine, glutamine, cystine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. Isolation of leucine with isoleucine (or vice versa), sequestering substitution of aspartic acid with glutamic acid (or vice versa), sequestering substitution of threonine with serine (or vice versa), or structurally of certain amino acids It is reasonably speculated that conservative substitutions with related amino acids will not significantly affect biological activity. Thus, a protein having substantially the same amino acid sequence as the corresponding molecule but having small amino acid substitutions that do not substantially affect the immunogenicity of the protein is within the defined range of the corresponding polypeptide.

The term epitope is the portion of a macromolecule that is recognized by the immune system and can induce a humoral type (B cell) and / or a cellular type (T cell) immune response. The term is also used interchangeably with “antigenic determinant” or “antigenic determinant site”. Antibodies that recognize the same epitope can be distinguished by simple immunoassays that show the ability of one antibody to block the binding of another antibody to a target antigen.
An “immunological response” to a composition or vaccine is the development of a cell-mediated and / or antibody-mediated immune response to the composition or vaccine in question in the host. Usually, an “immunological response” includes, but is not limited to, one or more of the following actions: to one or more antigens included in the composition or vaccine in question Production of specific antibodies, B cells, helper T cells and / or cytotoxic T cells. Preferably, the host will exhibit a therapeutic or protective immunological response so that resistance to new infections is enhanced and / or the clinical severity of the disease is reduced. Such protection may be presented by the reduction or elimination of symptoms usually exhibited by the infected host, the reduction of recovery time, and / or the reduction of viral titer in the infected host.

As used herein, an “immunogenic” protein or polypeptide also means an amino acid sequence that elicits an immunological response as described above. As used herein, an “immunogenic” protein or polypeptide includes the full length sequence of the protein, an analog thereof, or an immunogenic fragment thereof. By “immunogenic fragment” is meant a fragment of a protein that contains one or more epitopes and thus elicits an immunological response as described above. Such fragments can be identified using a number of epitope mapping techniques known in the art. See, for example: Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996). For example, a linear epitope can be synthesized, for example, by simultaneously synthesizing a large number of peptides on the solid phase (the peptide corresponds to a portion of the protein molecule), It can be determined by reacting with an antibody. Such techniques are known in the art and are described, for example, in: US Pat. No. 4,708,871; Geysen et al. 1984; Geysen et al. 1986 (all of which are hereby incorporated by reference in their entirety). include). Similarly, conformational epitopes can be readily identified by determining the spatial shape of amino acids, for example by X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, the above publication (Epitope Mapping Protocols). Methods particularly applicable to T. parva proteins are described in detail in PCT application PCT / US2004 / 022605, which is hereby incorporated by reference.
Synthetic antigens such as polyepitopes, flanking epitopes and other recombinant or synthetically derived antigens are also included in this definition. For example, see the following references (Bergmann et al. 1993; Bergmann et al. 1996; Suhrbier, 1997; Gardner et al. 1998). An immunogenic fragment for the purposes of the present invention will usually have at least about 3 amino acids, preferably at least about 5 amino acids, more preferably at least about 10-15 amino acids, most preferably about 15-25 of the molecule. Of amino acids or more. There is no critical upper limit on the length of the fragment, and the fragment will include a nearly full length protein sequence or even a fusion protein comprising at least one epitope of the protein.

Thus, the minimal structure of a polynucleotide that expresses an epitope comprises, consists essentially of, or consists of a nucleotide that encodes an epitope or antigenic determinant of an influenza protein or polyprotein. A polynucleotide encoding a whole protein or a fragment of a polyprotein is more advantageously at least 15 nucleotides, advantageously about 30-45 nucleotides, preferably about 45-75, preferably at least about the sequence encoding the whole protein or polyprotein. Contains, consists essentially of or consists of 57, 87 or 150 contiguous or adjacent nucleotides. Epitope determination methods such as the generation of overlapping peptide libraries (Hemmer et al. 1998), Pepscan (Geysen et al., 1984; Geysen et al., 1985; Van der Zee R. et al., 1989; Geysen, 1990; Multipin.RTM. Peptide Synthesis Kits de Chiron) and algorithms (De Groot et al., 1999) (PCT Application No. PCT / US2004 / 022605), both of which are hereby incorporated by reference in their entirety. Can be used for carrying out the present invention without requiring a complicated experiment. Other descriptions cited herein and further incorporated by reference may also be referenced for methods of determining epitopes of immunogens or antigens, and thus nucleic acid molecules encoding such epitopes.
“Polynucleotide” is a polymeric form of any length of nucleotides, including any combination of deoxyribonucleotides, ribonucleotides and analogs. A polynucleotide can have a three-dimensional structure and can perform any known or unknown function. The term “polynucleotide” includes double-stranded, single-stranded and triple-stranded helical molecules. Unless otherwise specified or required, any of the polynucleotide embodiments of the invention described herein are known or suspected to form double-stranded forms of DNA, RNA or hybrid molecules and said double-stranded forms. Each of the two complementary forms.

  The term “codon optimization” refers to the process of optimally constructing a nucleic acid sequence encoding a protein, polypeptide, antigen, epitope, domain or fragment for expression / translation in a selected host. In general, the expression level of a gene depends on many factors, such as promoter sequences and regulatory elements. One of the most important factors is the adaptation of the transcriptional gene codon usage to the typical codon usage of the host (G. Lithwich and H. Margalit, Genome Res. 2003, 13: 2665-2673). Thus, under translation selection, highly expressed genes in the prokaryotic genome have a significant codon usage bias. This is why they use a small codon subset that is recognized by the most abundant tRNA species (T. Ikemura, J Mol Biol, 1981, 151: 389-409). This ability to regulate codon adaptation is called translational selection and its strength is important in rapidly growing bacteria (Rocha, EP, Genome Res. 14, 2279-2286, 2004; Sharp, PM et al., Nucleic Acids Res. 33, 1141-1153). If the gene contains codons that the host rarely uses, its expression level will not be maximal. This is due to the expression of heterologous proteins (Gustafsson, C. et al., Trends Biotechnol. 22, 346-353, 2004) and DNA vaccines (C. Ivory and K. Chadee, Genet. Vaccines Ther. 2, 17, 2004). It can be one of the limits. A large number of synthetic genes have been redesigned to increase their expression levels. The Synthetic Gene Database (SGDB) (G. Wu et al., Nucleic Acids Res. 35, D76-D79, 2007) contains information on over 200 published experiments on synthetic genes. In the process of designing a nucleic acid sequence that is inserted into a new host to express an optimal amount of a particular protein, optimizing codon usage is usually one of the first steps (C. Gustafsson, Trends Biotechnol. 22, 346-353, 2004). Optimizing codon usage basically involves changing rare codons in the target gene to more closely reflect the host's codon usage without altering the amino acid sequence of the encoded protein. Necessary (Gustafsson, C., Trends Biotechnol. 22, 346-353, 2004). Thus, the information normally used for the optimization process is the DNA or protein sequence to be optimized and the host codon usage table (reference set).

  There are several public web servers and independently usable applications that allow some codon optimization by those skilled in the art: 'GeneDesign' (SM Richardson et al. Genome Res. 16, 550-556, 2006) ), 'Synthetic Gene Designer' (G. Wu et al. Protein Expr. Purif. 47, 441-445, 2006) and 'Gene Designer' (A. Villalobos et al. BMC Bioinformatics 7, 285, 2006) A package that provides a platform for gene design (including codon optimization steps). Regarding the method for codon usage optimization available in each server or program, the first program developed used only the '1 amino acid-1 codon' approach. More recent programs and servers include yet another way to create some degree of codon usage diversity. This diversity reflects the codon usage diversity of the naturally highly expressed genes and allows the introduction of additional criteria (eg, avoidance of restriction sites) in the optimization process. Most applications and web servers described herein provide the following three codon optimization methods: full optimization of all codons, relative codon usage of the reference set using the Monte Carlo approach And a new approach designed to maximize optimization while minimizing changes between query and optimized sequences. In certain embodiments herein, the sequence encoding the protein complement of NDV is a codon optimized for avian cell expression, and in a preferred embodiment, nucleic acids encoding NDV P, L and NP. The sequence is a codon optimized for expression in avian cells. In yet another embodiment, the nucleic acid sequence encoding the recombinant protein, antigen, peptide, polypeptide, fragment, domain or epitope is a codon optimized for expression in birds. In another embodiment, the codon optimized sequence encodes an avian expression AIV protein, antigen, peptide, polypeptide, fragment, domain or epitope. In yet another embodiment, the codon optimized sequence encodes the AIV HA and / or N protein, antigen, peptide, polypeptide, fragment, domain or epitope for avian expression.

The following are non-limiting examples of polynucleotides: gene or gene fragment, exon, intron, mRNA, tRNA, rRNA, siRNA, ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, any sequence Isolated DNA, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can include modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracil, other sugars and linking groups (eg, fluororibose and thiolate), and nucleotide branches. The nucleotide sequence can be modified after polymer formation, for example by ligation, using a labeling component. Other types of modifications in this definition include caps, substitution of one or more naturally occurring nucleotides by analogs, and placing the polynucleotide on a protein, metal ion, labeling component, other nucleotide or solid phase. The introduction of means for coupling is included. The polynucleotide can be obtained by chemical synthesis or derived from a microorganism.
The invention further includes complementary strands of polynucleotides encoding influenza proteins, antigens, epitopes or immunogens. The complementary strand can be a polymer of any length and can be an analog of deoxyribonucleotides, ribonucleotides and any combination of the foregoing.
The terms “protein”, “peptide”, “polypeptide” and “polypeptide fragment” are used interchangeably herein and refer to a polymer of amino acid residues of any length. The polymer may be linear or branched, may contain modified amino acids or amino acid analogs, and may be interrupted by chemical components other than amino acids. The term may also be modified naturally or by intervention, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (eg, linking with a labeling component or biologically active component). Of the amino acid polymer formed.

An “isolated” polynucleotide or polypeptide is one that is substantially free of the materials associated with it in its natural state. Substantially absent means that at least 50%, at least 70%, at least 80%, at least 90% or at least 95% of these materials are absent.
Hybridization reactions can be performed under various “stringency” conditions. Conditions for increasing the stringency of a hybridization reaction are well known. See, for example, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al., 1989). Examples of relevant conditions include (listed in order of increasing stringency): 25 ° C, 37 ° C, 50 ° C and 68 ° C incubation temperatures; 10 x SSC, 6 x SSC, 1 x SSC, 0.1 × SSC buffer concentration (SSC is 0.15 M NaCl and 15 mM citrate buffer) and equivalents as described above using other buffer systems; 0%, 25%, 50% and 75% formamide concentrations; 5 to 24 hours incubation time; 1, 2 or 3 or more washing steps; 1, 2 or 15 minutes washing incubation time; and 6 x SSC, 1 x SSC, 0.1 x SSC, or deionized water wash solution .

  The present invention further provides for polynucleotides encoding functionally equivalent variants and derivatives of influenza polypeptides and functionally equivalent fragments thereof (and thus significantly affecting the intrinsic properties of the polypeptides encoded by them). Can be strengthened or attenuated without affecting These functionally equivalent variants, derivatives and fragments exhibit the ability to maintain influenza activity. For example, changes in DNA that do not change the encoded amino acid sequence are similar to changes that result in conservative substitution of amino acid residues, deletion or addition of one or several amino acids, and substitution of amino acid residues by amino acid analogs. In addition, changes that do not significantly affect the properties of the encoded polypeptide. Conservative amino acid substitutions are glycine / alanine; valine / isoleucine / leucine; asparagine / glutamine; aspartic acid / glutamic acid; serine / threonine / methionine; lysine / arginine; and phenylalanine / tyrosine / tryptophan. In certain embodiments, the variant is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least with the influenza polynucleotide or polypeptide of interest. 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% Or at least 99% homology or identity.

In one aspect, the invention provides influenza polypeptides, specifically avian influenza polypeptides. In another aspect, the invention provides a polypeptide having the sequence shown in SEQ ID NO: 15, 17, 19, 21, or 23, and a variant or fragment as described above.
In another aspect, the invention provides a tri-HA polypeptide of the invention, specifically a polypeptide having the sequence shown in SEQ ID NO: 15, 17, 19, 21, or 23 and at least 70%, at least 75%, Polypeptides having at least 80%, at least 85%, at least 90%, at least 95%, 96%, 97%, 98% or 99% sequence identity are provided.
In yet another aspect, the present invention facilitates fragments and variants of influenza polypeptides identified above (SEQ ID NO: 15, 17, 19, 21, or 23) and variants (using well-known molecular biology techniques) Can be prepared).
Variants include antigenic polypeptides of the invention, specifically the amino acid sequence set forth in SEQ ID NO: 15, 17, 19, 21 or 23 and at least about 75%, 80%, 85%, 90%, 95%, 96 A homologous polypeptide having an amino acid sequence of%, 97%, 98% or 99% identity.
An immunogenic fragment of an influenza polypeptide is at least 8, 10, 15, or 20 consecutive influenza polypeptides having a sequence set forth in SEQ ID NO: 2, 4, 5, 8, 10, 12, or 14 or variants thereof Contains amino acids, at least 21 amino acids, at least 23 amino acids, at least 25 amino acids or at least 30 amino acids. In another embodiment, the influenza polypeptide fragment comprises a specific antigenic epitope found in the full-length influenza polypeptide.

In another aspect, the invention provides a polynucleotide encoding a polynucleotide encoding an influenza HA polypeptide, eg, a polypeptide having the sequence set forth in SEQ ID NO: 15, 17, 19, 21, or 23. In yet another aspect, the invention provides at least 70%, at least 75%, at least 80%, at least 85%, at least 90% with a polypeptide having the sequence shown in SEQ ID NO: 15, 17, 19, 21, or 23, Polypeptides having at least 95%, 96%, 97%, 98% or 99% sequence identity, or conservative variants, allelic variants, homologues, or at least 8 or at least 10 consecutive ones of these polypeptides Provided are polynucleotides encoding immunogenic fragments comprising amino acids, or combinations of these polypeptides. A polynucleotide encoding an influenza HA polypeptide can be codon optimized for expression in a particular animal species. The HA protein can be modified at the cleavage site from a highly pathogenic avian influenza sequence (multiple basic amino acids: RERRRKKR—SEQ ID NO: 25) to a low pathogenic avian influenza sequence (RETR—SEQ ID NO: 26).
In another aspect, the present invention provides a polynucleotide having the nucleotide sequence set forth in SEQ ID NOs: 14, 16, 18, 21, 22, or variants thereof. In yet another aspect, the invention provides at least 70%, at least 75%, at least 80%, at least 85%, at least 90% of one of the polynucleotides having the sequence shown in SEQ ID NO: 14, 16, 18, 21, 22. A polynucleotide or variant thereof having%, at least 95%, 96%, 97%, 98% or 99% sequence identity is provided.

For purposes of the present invention, sequence identity or homology is determined by comparing sequences when performing alignments with minimal sequence gaps while maximizing overlap and identity. In particular, sequence identity can be determined using any of a number of mathematical algorithms. A non-limiting example of a mathematical algorithm used to compare two sequences is the algorithm of Karlin et al. (1990) modified by Karlin et al. (1993).
Another example of a mathematical algorithm used for sequence comparison is the algorithm of Myers et al. (1988). Such an algorithm is the ALIGN program (version 2.0) (which is part of the GCG sequence alignment software package). When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Yet another algorithm useful for the identification and alignment of regions with local sequence homology is the FASTA algorithm described by Pearson et al. (1988).
In general, amino acid sequence comparison is accomplished by aligning the amino acid sequence of a polypeptide of known structure with the amino acid sequence of a polypeptide of unknown structure. Subsequently, the amino acids of the sequences are compared and a group of homologous amino acids is put into the group together. By this method, conserved regions of the polypeptide are detected and amino acid insertions and deletions are revealed. Homology between amino acid sequences can be determined by using commercially available algorithms (see also above description on homology). In addition to those described elsewhere herein, BLAST, Gapped BLAST, BLASTN, BLASTP, and PSI-BLAST programs (provided by the National Center for Biotechnology Information) are also described. These programs are widely used for this purpose in the art and can perform alignment of homologous regions of two amino acid sequences.

In any set of search programs, the gapped alignment routine is integral to the database search itself. Gap addition can be turned off if desired. The default penalty (Q) for a gap of length 1 is Q = 9 for proteins and BLASTP and Q = 10 for BLASTN, but can be changed to any integer. The default penalty (R) per residue for gap extension is R = 2 for proteins and BLASTP and R = 10 for BLASTN, but can be changed to any integer. Any combination of Q and R values can be used to perform sequence alignment to minimize sequence gaps while maximizing overlap and identity. The default amino acid comparison matrix is BLOSUM62, but other amino acid comparison matrices (eg, PAM) can also be used.
Alternatively or additionally, the term “homology” or “identity”, for example with respect to nucleotide or amino acid sequences, can indicate a quantitative measure of homology between two sequences. Percent sequence identity can be calculated as (N _ref −N _dif ) × 100 / N _ref . _Where N _dif is the total number of residues that are not identical when alignment is performed, and N _ref is the number of one residue in the sequence. Thus, the DNA sequence AGTCAGTC will have 75% sequence identity with the sequence AATCAATC (N _ref = 8; N _dif = 2).
Apart from or in addition to the above, “homology” or “identity” with respect to a sequence is a position where the nucleotides or amino acids divided by the number of nucleotides or amino acids in the shorter sequence of the two sequences are identical Can mean a number of. In this case, the alignment of the two sequences is performed according to Wilbur and Lipman's algorithm (Wilbur et al. 1983, which is incorporated herein by reference), for example, a 20 nucleotide window size, a 4 nucleotide word length. And 4 gap penalties, as well as computer-aided analysis, and interpretation of sequence data, including alignments, should be done conveniently using commercially available programs (eg Vector NTI Software ™, Invitrogen Inc. CA, USA) Can do. When the RNA sequence is similar to the DNA sequence or has some sequence identity or homology, the thymidine (T) in the DNA sequence is considered equivalent to the uracil (U) of the RNA sequence. Thus, RNA sequences are within the scope of the present invention and can be derived from DNA sequences by regarding the DNA sequence thymidine (T) as equivalent to the RNA sequence uracil (U).
Furthermore, without performing tedious experiments, one skilled in the art can refer to many other programs or references for the determination of percent homology.

The invention further encompasses influenza polynucleotides that are included in vector molecules or expression vectors, and further operably linked to promoter elements and optionally enhancers.
“Vector” means a recombinant DNA or RNA plasmid, bacteriophage, or virus that contains a heterologous polynucleotide to be delivered to a target cell in vitro or in vivo. The heterologous polynucleotide can include the sequence of interest for the purposes or treatment of the present invention, and can optionally have the form of an expression cassette. As used herein, a vector need not have replication capacity in the final target cell or target subject. The term includes cloning vectors as well as viral vectors.
The term “engineered” or “recombinant” means a polynucleotide of semi-synthetic or synthetic origin that is not naturally occurring or linked to another polynucleotide in an organization not found in the natural state. To do.
“Heterologous” means that an entity is derived from an entity that is genetically distinct from the rest of the entity being compared. For example, a polynucleotide can be incorporated by genetic engineering techniques into a plasmid or vector derived from a different source, and the polynucleotide is thus a heterologous polynucleotide. A promoter removed from its original coding sequence and operably linked to a coding sequence other than the original sequence is a heterologous promoter.

The polynucleotides of the present invention may comprise additional sequences, such as additional coding sequences within the same transcription unit, control elements (eg, promoters), ribosome binding sites, 5′UTR, 3′UTR, transcription terminators, polyadenylation sites, the same or Includes additional transcription units under the control of different promoters, sequences that allow cloning, expression, homologous recombination and host cell transformation, and any constructs that may be desirable to provide embodiments of the invention be able to.
Advantageously, elements for expressing influenza polypeptides, antigens, epitopes or immunogens are present in the vectors of the invention. In a minimal aspect, the above includes initiation codons (ATG), stop codons and promoters, and optionally polyadenylation for certain vectors (eg, plasmids and certain viral vectors, eg, viral vectors other than poxviruses). Comprise, consist essentially of, or consist of the above. When the polynucleotide encodes a polyprotein fragment (eg, an influenza peptide), advantageously in a vector, the ATG is located 5 ′ of the reading frame and the stop codon is located 3 ′. Other components that control expression may also be present. Said other components are e.g. enhancer sequences, stabilizing sequences, e.g. introns and / or untranslated 5 'or 3' sequences, and signal sequences allowing the secretion of the protein.

  Methods for making and / or administering vectors or recombinants or plasmids for in vivo or in vitro expression of the gene product of the gene of the invention are disclosed in any desired method, such as the following references: Or the methods disclosed in the references cited in the following references or similar methods: US Pat. Nos. 4,603,112; 4,769,330; 4,394,448; 4,722,848; 4,745,051; 4,769,331; 4,945,050; 5,494,807 No. 5,514,375; 5,744,140; 5,744,141; 5,756,103; 5,756,103; 5,762,938; 5,766,599; 5,990,091; 5,174,993; 5,505,941; 5,338,683; 5,494,807; 6,580,859; 6,130,066; 6,004,777; 6,130,066; 6,497,883; 6,464,984; 6,451,770; 6,391,314; 6,387,376; 6,376,473; 6,368,603; 6,348,196; No. 62; 6,217,883; 6,207,166; 6,207,165; 6,207,165; 6,159,477; 6,153,199; 6,090,393; 6,074,649; 6,045,803; 6,033,670; United States Patent Application 920,197 (filed October 16, 1986); WO 90/01543; W091 / 11525; WO 94/16716; WO 96/39491; WO 98/33510; EP 265785; EP 0 370 573; Andreansky et Ballay et al., 1993; Felgner et al., 1994; Frolov et al., 1996; Graham, 1990; Grunhaus et al., 1992; Ju et al., 1998; Kitson et al., 1991 McClements et al., 1996; Moss, 1996; Paoletti, 1996; Pennock et al., 1984; Richardson (Ed), 1995; Smith et al., 1983; Robertson et al., 1996; Robinson et al., 1997 And Roizman, 1996. The documents cited herein and incorporated herein by reference not only provide examples of vectors useful in the practice of the invention, but also include vectors of the compositions of the invention or compositions of the invention. Can provide a source for non-influenza peptides or fragments thereof to be expressed by the vectors contained therein.

The present invention also relates to a preparation comprising a vector (eg, an expression vector), such as a prophylactic or therapeutic composition. Said preparation comprises, consists essentially of or consists of (or more advantageously expresses) one or more influenza polypeptides, antigens, epitopes or immunogens Or may comprise, consist essentially of, or consist of two or more vectors, such as expression vectors (eg, in vivo expression vectors). The vector comprises a polynucleotide encoding (and more advantageously expressing) an influenza antigen, epitope or immunogen in a pharmaceutically or veterinarily acceptable carrier, excipient or vehicle and further expressing said To do. Thus, according to an embodiment of the invention, the other vector or vectors in the preparation constitute an influenza polypeptide, antigen, epitope or immunogen (eg hemagglutinin, neuraminidase, nucleoprotein) or a fragment thereof. The vector comprises, consists essentially of, or consists of the above, encoding a polynucleotide encoding one or more other proteins, and the vector expresses them in an appropriate environment.
According to another embodiment, the vector or vectors in the preparation comprise a polynucleotide encoding one or more proteins or fragments thereof of an influenza polypeptide, antigen, epitope or immunogen. Or consisting essentially of or consisting of said vector, wherein said vector or vectors express said polynucleotide. The preparation of the invention comprises, consists essentially of or consists of at least two vectors, said vectors being derived from different influenza isolates and encoding the same protein and / or different proteins The polynucleotide comprises, consists essentially of or consists of the foregoing, and is further advantageously expressed in vivo under reasonable conditions or under appropriate conditions or in a suitable host cell. The preparation comprises, consists essentially of, or consists of a polynucleotide encoding an influenza polypeptide, antigen, fusion protein or epitope thereof, and more advantageously expressing it in vivo. The invention also includes different influenza proteins, polypeptides, antigens, epitopes or immunogens such as humans, horses, pigs, seals, whales in addition to different species (eg, bird species including chicken, turkey, duck and cancer) Intended to be a mixture of vectors comprising, consisting essentially of or consisting of influenza polypeptides, antigens, epitopes or immunogens derived from, but not limited to.

The term plasmid includes any transcription unit comprising a polynucleotide of the invention and the elements necessary for said in vivo expression in a cell or in a desired host or target cell, and in this regard, a supercoil. It should be noted that the plasmid and all its topological isomers, open circular plasmids, as well as linear plasmids, are included within the scope of the present invention.
Each plasmid is optionally fused to a polynucleotide encoding a heterologous peptide sequence, variant, analog or fragment and optionally a polynucleotide encoding a recombinant protein, antigen, epitope or immunogen and is operably linked to a promoter. Contain, contain, or consist essentially of anything that is or is under the control of or is dependent on the promoter. In general, it is advantageous to utilize a strong promoter that functions in eukaryotic cells. A preferred strong promoter is the immediate early cytomegalovirus promoter (CMV-IE) of human or murine origin and possibly other origins (eg from rat or guinea pig). The CMV-IE promoter may be an actual promoter segment, but may or may not be accompanied by an enhancer segment. Reference may be made to the following documents: EP-A-260 148; EP-A-323 597; US Pat. Nos. 5,168,062, 5,385,839 and 4,968,615; and PCT application WO 87/03905. The CMV-IE protein is advantageously human CMV-IE (Boshart et al., 1985) or murine CMV-IE.

In a more general relationship, the promoter is of viral or cellular origin. Strong viral promoters other than CMV-IE that may be useful in the practice of the present invention are the early / late promoters of the SV40 virus or the LTR promoter of Rous sarcoma virus. Strong cellular promoters that may be useful in the practice of the present invention are cytoskeletal gene promoters, such as the desmin promoter (Kwissa et al., 2000) or the actin promoter (Miyazaki et al., 1989).
Functional subfragments of these promoters (ie, portions of these promoters that maintain proper facilitating activity) are also included within the scope of the present invention, eg, the truncated CMV-IE promoter of PCT application WO98 / 00166 or US Pat. No. 6,156,567. Can be used in the practice of the present invention. Thus, the promoter used in the practice of the present invention has the activity of the actual or full length promoter from which the derivative or subfragment was derived, preferably maintaining the appropriate promoting activity and thus functioning as a promoter. Derivatives and subfragments of the full-length promoter that are substantially similar (eg, similar to the activity of the truncated CMV-IE promoter of US Pat. No. 6,156,567 to full-length CMV-IE promoter activity) are included. Thus, the CMV-IE promoter used in the practice of the present invention comprises, or consists essentially of, the promoter portion of the full-length promoter and / or the enhancer portion of the full-length promoter as well as derivatives and subfragments, Or consisting of the above.
Preferably, the plasmid comprises or essentially consists of other expression control elements. It is particularly advantageous to incorporate a stabilizing sequence such as an intron sequence, preferably hCMV-IE (PCT application WO89 / 01036), intron II of the rabbit β globulin gene (van Ooyen et al. 1979).
For polyadenylation signals (polyA) for viral vectors other than plasmids and poxviruses, the poly (A) signal of the bovine growth hormone (bGH) gene (see US Pat. No. 5,122,458) or the rabbit β globulin gene Poly (A) or SV40 viral poly (A) signals may be useful.

According to another embodiment of the invention, the expression vector is an expression vector used for in vitro expression of the protein in a suitable cell line. The expressed protein is collected in or from the culture supernatant after or prior to secretion (if not secreted, typically cell lysis occurs or is performed), optionally For example, it is concentrated by a concentration method such as ultrafiltration and / or purified by a purification means such as affinity, ion exchange or gel filtration chromatography.
“Host cell” means a prokaryotic or eukaryotic cell that has been genetically modified or can be genetically modified by administration of an exogenous polynucleotide, such as a recombinant plasmid or vector. When referring to genetically modified cells, the term refers to both the originally modified cell or its progeny cells. Preferred host cells include baby hamster kidney (BHK) cells, colon cancer (Caco-2) cells, COS7 cells, MCF-7 cells, MCF-10A cells, Madin-Darby canine kidney (MDCK) strains, mink lungs ( Mv1Lu) cells, MRC-5 cells, U937 cells and VERO cells, but are not limited to these. A polynucleotide containing the desired sequence can be inserted into a suitable cloning or expression vector, and this vector can be sequentially introduced into a suitable host cell for replication and amplification. The polynucleotide can be introduced into the host cell by any means known in the art. A vector containing the polynucleotide of interest can be introduced into the host cell by any number of suitable means. Such means include direct uptake; endocytosis; transfection; F-conjugation; electroporation; transfection utilizing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; microprojectile bombardment; Lipofection; and infection (for example, a retroviral vector if the vector is infectious). The choice of vector or polynucleotide introduction will often depend on the characteristics of the host cell.

In one embodiment of the invention, the vector is a Newcastle disease virus (NDV) vector. Newcastle disease virus, also called avian paramyxovirus 1 (APMV1, Paramyxoviridae, Paramyxovirinae, Avravirus), is an avian pathogen, and its naturally occurring strain exhibits widespread disease severity . NDV is particularly advantageous as a veterinary vaccine vector. This is because the vector itself functions as a required poultry vaccine. The pathogenic forms of NDV strains are asymptomatic intestinal type (eg Ulster 2C, Queensland V4), roentogenic type (eg Hitchner B1, F (eg Asplin), La Sota), mesogenic type (eg H, Mukteswar, Rakin, Beaudette C strain) or velogenic type (eg Texas GB, NY parrot 70181, Italien, Milano, Herts 33/56). Advantages of influenza vector vaccines based on NDV vectors include, but are not limited to: (1) induce a wide range of immunity including humoral, cellular and mucosal responses; (2) NP and M do not express the protein, thus conforming to the DIVA (identification of infected animals from the vaccine immunized animals (d ifferentiate i nfected from v accinated a nimals)); induce (3) rapid immune initiation; (4) (5) In case of accidental release, there is less production risk to the environment than inactivated vaccines.

  Certain characteristics of NDV suggest that recombinant NDV (rNDV) or engineered NDV expressing foreign viral proteins would be very good vaccine candidates. NDV grows to very high titers in many cell lines and eggs and induces strong humoral and cellular immune responses in vivo. NDV naturally infects mucosal surfaces of the respiratory and esophagus and is therefore particularly useful for the delivery of protective antigens of respiratory disease pathogens (eg AIV). Furthermore, commercially available live NDV vaccines are widely used in the United States and most other countries. Vaccines based on live NDV recombinants may also have advantages over other recombinant live vaccine vectors. First, foreign proteins are expressed with only a few NDV proteins. In contrast, poxvirus and herpesvirus vectors express numerous other proteins derived from their large size genomes. Where a specific immune response occurs with the application of a vaccine, it may be advantageous to express a limited number of proteins. Second, NDV replicates in the cytoplasm of infected cells without the DNA phase, which eliminates the problem of integrating the viral genome into host cell DNA. This virus does not undergo detectable genetic recombination.

Another application of reverse genetics is the development of more effective and superior NDV vaccines as described herein. Conventional NDV vaccines utilize naturally occurring lentogenic strains (eg, Hitchner B1 and LaSota). As can be seen with other live attenuated vaccines, conventional NDV vaccines can still cause illness to revert to virulence. The selection of the NDV vaccine strain as a vector is important. This is because the first data reported for the Hitchner B1 NDV strain was disappointing. The data obtained later on the LaSota NDV strain was reliable. Vaccine candidates based on this strain are used outdoors in China and Mexico.
The invention herein discloses the applicant's use of the NDV AVINEW ™ vaccine strain as a vector for delivery of recombinant-derived antigens to birds. More specifically, the antigen can be an influenza virus (AIV) antigen. AVINEW (TM) is a live NDV vaccine developed by Merial Ltd. and used worldwide. This vaccine strain is naturally non-pathological and exhibits a respiratory and intestinal duality. This branched tropism in birds is inherent to recombinant NDV (rNDV) or engineered NDV vaccines that utilize the AVINEW ™ backbone for gene delivery in birds, with respect to interference with maternally derived antibodies (MDA) against NDV itself. Biological characteristics can be given. Such resistance to MDA is inherently possessing significant levels of NDV-specific MDA or may be resistant to more common recombinant NDV immunity from commercially available birds and wild type Active immunization of birds can be enabled.
Furthermore, the AVINEW ™ strain belongs to the NDV gene group or type (class 2, genotype I) that can infect ducks. In contrast to LaSota, whose tropism is essentially airway oriented, the AVINEW ™ strain does not induce respiratory adverse reactions.

In certain embodiments, the NDV vector is NDV AVINEW ™. The NDV vector may also be the vector of US Pat. No. 5,118,502, specifically the strain deposited as ATCC No. VR2239.
One embodiment of the invention provides the genomic DNA sequence of AVINEW and the encoded protein sequence. The genomic DNA sequence of the AVINEW NDV strain has the polynucleotide sequence shown in SEQ ID NO: 1. The AVINEW genomic cDNA sequence (SEQ ID NO: 1) is different from the VG / GA sequence (GenBank Accession No. EU289028). The sequence identity between the genomic sequences of AVINEW (SEQ ID NO: 1) and VG / GA (EU289028) is 89.6%. The amino acid sequence identity between the proteins of the Avinew strain and the VG / GA strain is 95.9% for the NP protein (Avinew SEQ ID NO: 3 and GenBank No. ABZ80386), and the P protein (Avinew SEQ ID NO: 5 for GenBank). No. ABZ80387) 89.7%, M protein (Avinew SEQ ID NO: 7 against GenBank No. ABZ80388) 94.2%, F protein (Avinew SEQ ID NO: 9 against GenBank No. ABZ80389) 92.4%, 88.1% for the HN protein (GenBank No. ABZ80390 for Avinew SEQ ID NO: 11) and 96.9% for the L protein (GenBank No. ABZ80391 for Avinew SEQ ID NO: 13). Nucleic acid sequence identity between the Avinew and VG / GA strain genes is 90.6% for the NP gene (Avinew SEQ ID NO: 2 to 122-1591 bp of EU289028), P gene (Avinew SEQ ID NO: 4 On the other hand, 88.6% for EU289028 1887-3074bp), 90.1% for M gene (Avinew SEQ ID NO: 6 vs EU289028 3290-4384bp), F gene (Avinew SEQ ID NO: 8 vs EU289028 94.7% for 4544-6205 bp), 84.7% for HN gene (6412-8145 bp of EU289028 for Avinew SEQ ID NO: 10), and 8381-14995 bp of EU289028 for SEQ ID NO: 12 of Avinew ) Is 90.9%. Comparison of the complete genomic sequence and individual gene sequences with other available NDV reference sequences indicates that the AVINEW NDV strain is genetically related to Australian lentogenic strains 98-1154 and Queensland V14. It was done. The complete genomic sequence of NDV 98-1154 is shown in GenBank AY935491. The sequence identity between the genomic sequences of AVINEW (SEQ ID NO: 1) and AY935491 is 98.8%. The amino acid sequence identity between the proteins of Avinew strain and VG / GA strain is 99.8% for NP protein (Genbank No. AAX45376 against SEQ ID NO: 3 for Avinew) and P protein (for SEQ ID NO: 5 for Avinew). GenBank No. AAX45377) is 97.7%, M protein (Avinew SEQ ID NO: 7 against GenBank No. AAX45378) is 98.4%, F protein (Avinew SEQ ID NO: 9 against GenBank No. AAX45379) 98.7% for HN protein (Generic No. AAX45380 for Avinew SEQ ID NO: 11) and 99.5% for L protein (GenBank No. AAX45381 for Avinew SEQ ID NO: 13) is there. The nucleic acid sequence identity between the genes of Avinew strain and VG / GA strain is 99.0% for NP gene (Avinew SEQ ID NO: 2 to 122-1591 bp of AY935491), P gene (Avinew SEQ ID NO: 4 On the other hand, 98.2% for AY935491 1887-3074bp), 98.6% for M gene (Avinew SEQ ID NO: 6 3290-4384bp AY935491), F gene (Avinew SEQ ID NO: 8 AY935491 48.8-6205 bp) for 98.8%, HN gene (Avinew SEQ ID NO: 10 for AY935491 6412-8154 bp) for 93.1%, and L gene (Avinew SEQ ID NO: 12 for AY935491 8381-14995 bp) ) Is 98.8%. For Queensland V4, only partial sequences are available in GenBank.

In another embodiment, the present invention provides a polynucleotide having the sequence shown in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, or 24 and variants or fragments thereof. The invention further includes complementary strands of the polynucleotides disclosed herein. In yet another embodiment, the present invention provides a polypeptide having the sequence shown in SEQ ID NO: 3, 5, 7, 9, 11 or 13 and variants or fragments thereof.
In another aspect, the present invention provides a genomic cDNA of AVINEW having the sequence shown in SEQ ID NO: 1 or 24. In yet another embodiment, the polynucleotide is the reverse complementary strand of a polynucleotide having the sequence shown in SEQ ID NO: 1 or 24. In yet another embodiment, a polynucleotide of the invention or its reverse complement is at least 70%, at least 75%, at least 80%, at least 85%, at least with a polynucleotide having the sequence shown in SEQ ID NO: 1 or 24 It has 90%, at least 95%, 96%, 97%, 98% or 99% sequence identity.
In certain embodiments, the invention provides a fragment of a polynucleotide that encodes a polynucleotide that encodes an AVINEW polypeptide, eg, a polypeptide having the sequence shown in SEQ ID NO: 3, 5, 7, 9, 11, or 13. To do. In yet another aspect, the invention provides at least 70%, at least 75%, at least 80%, at least 85%, at least 90% of a polypeptide having the sequence shown in SEQ ID NO: 3, 5, 7, 9, 11 or 13. Polypeptides having%, at least 95%, 96%, 97%, 98% or 99% sequence identity, or conservative variants, allelic variants, homologues or at least 8 or at least 10 consecutive ones of these polypeptides Provided are polynucleotides that encode immunogenic fragments comprising amino acids, or combinations of these polypeptides.

In another aspect, the invention provides a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, or 24, or a variant thereof. In yet another embodiment, the polynucleotide is the reverse complement of a polynucleotide having the sequence shown in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, or 24. In yet another aspect, the invention provides at least 70%, at least 75%, at least, a polynucleotide having one of the sequences shown in SEQ ID NOs: 1, 2, 4, 6, 8, 10, 12, or 24 or one of its variants. Provided are polynucleotides or reverse complements thereof having 80%, at least 85%, at least 90%, at least 95%, 96%, 97%, 98% or 99% sequence identity.
In one aspect, the invention relates to a pharmaceutical composition or vaccine that induces an immunological response in a host animal inoculated with the vaccine or composition, wherein the composition comprises a pharmaceutically acceptable carrier and a modified AVENEW recombinant virus. including. In yet another aspect of the invention, the engineered AVINEW virus comprises a heterologous DNA sequence encoding an antigenic protein from a pathogen in a non-essential region of the viral genome, and the vaccine is administered when administered to a host. An immunological response specific to the protein encoded by the pathogen can be induced.

The term “non-essential region” refers to a region of the viral genome that is not essential for virus replication and propagation in tissue culture, and whose deletion or inactivation may reduce virulence in diverse animal systems. means. Any non-essential region or part thereof can be deleted from the AVINEW genome, or foreign sequences can be inserted therein, and the survival activity and stability of the engineered AVINEW resulting from the deletion or insertion is Can be used to confirm whether the deletion region or part thereof is actually non-essential. In another embodiment, the non-essential region of the AVINEW genome is a region between the P gene and the M gene, or a region between the M gene and the F gene of the AVINEW genome. In yet another embodiment, the non-essential region is in the region of 3075nt-3289nt or 4385nt-4543nt of SEQ ID NO: 1, 3115nt-3353nt or 4449nt-4607nt of SEQ ID NO: 24.
One aspect of the present invention relates to NDV vectors that express avian antigens. The antigen can be an avian influenza antigen. The avian influenza antigen can be a hemagglutinin, such as H5 HA.

NDV vectors expressing avian influenza genes, such as the following, are also contemplated for the present invention: wild type HA and HPAIV inserted into the intergenic region between the P and M genes of the LaSota NDV vaccine strain H5 subtype avian influenza virus (AIV) hemagglutinin (HA) with both mutant HA open reading frames derived from wild bird isolates (A / Bar-Headed goose / Qinghai / 3/2005 (H5N1)) NDV constructs that express E. coli (see, eg, Ge et al., Journal of Virology, Jan. 2007, 81 (1): 105-158); influenza virus deposited at GenBank with accession number AF375823 A complete cDNA clone of the Newcastle disease virus (NDV) vaccine strain Hitchner B1 that expresses (see, eg, Nakaya et al., Journal of Virology, Dec. 2001, pp. 11868-11873); HPAIV A / chicken / NDV recombinant (NDVH5Vm) expressing the H5 protein of Nam / P41 / 05 (H5N1) (see, eg, Roemer-Oberdoerfer et al., Vaccine. 2008 May 2; 26 (19): 2307-13 Epub 2008 Mar 18); A roentigenic paramyxovirus type 1 vector (NDV B1) with the hemagglutinin (HA) gene from avian influenza virus (AIV) A / chicken / NY / 13142-5 / 94 (H7N2) inserted RNDV-AIV-H7 constructed using (eg see below: Swayne et al., Avian Diseases 47: 1047-1050, 2003); H5 subtype NDV expression constructed by reverse genetics Avian influenza virus (AIV) hemagglutinin (see, for example, Veits et al., PNAS, May 23, 2006, 103 (21): 8197-8202); and based on the lentogenic NDV vaccine strain clone 30 Expresses the HA gene of AIV A / chicken / Vietnam / P41 / 2005 (H5N1) The recombinant NDV-H5 (see, for example, the following:. Veits et al, Vaccine (2008) 26: 1688-1696).
In yet another embodiment of the invention, an avian influenza virus that expresses the NDV gene, for example a chimeric avian influenza virus that expresses the ectodomain of the hemagglutinin-neuraminidase gene of NDV instead of the neuraminidase protein of H5N1 avian influenza virus, or fusogenic Bivalent vaccine expressing the ectodomain of H7 avian influenza virus hemagglutinin in the attenuated NDV gene (including only the ectodomain together with the transmembrane domain and cytoplasmic domain from the NDV F protein) (see below, for example) Please: Park et al., PNAS, May 23, 2006, 103 (21): 8203-8308) is also contemplated.

In certain embodiments, the present invention provides for the administration of a therapeutically effective amount of a formulation for delivery of protein, antigen, epitope or immunogen and expression in target cells. The determination of a prophylactically or therapeutically effective amount is routine to those of ordinary skill in the art. In another embodiment, the formulation comprises an expression vector comprising a polynucleotide that expresses an influenza antigen, epitope or immunogen and a pharmaceutically or veterinarily acceptable carrier, vehicle or excipient. In another embodiment, the pharmaceutically or veterinarily acceptable carrier, vehicle or excipient facilitates transfection and / or improves storage of the vector or protein.
Pharmaceutically or veterinary acceptable carriers, vehicles or excipients are well known to those skilled in the art. For example, a pharmaceutically or veterinarily acceptable carrier or vehicle or excipient can be sterile water, 0.9% NaCl (eg, saline) solution or phosphate buffer. Other pharmaceutically or veterinary acceptable carriers or vehicles or excipients that can be used in the methods of the present invention include, but are not limited to, poly- (L-glutamate) or polyvinylpyrrolidone. A pharmaceutically or veterinarily acceptable carrier or vehicle or excipient can be any compound or combination of compounds that facilitates administration of a vector (or a protein expressed in vitro from a vector of the invention). Advantageously, the carrier, vehicle or excipient may facilitate transfection and / or improve the storage of the vector (or protein). Dosages and dose volumes are discussed herein in the general description, and further upon reading this disclosure in conjunction with information in the field, one of ordinary skill in the art will be able to perform without any tedious experimentation. The above can be determined.

  Cationic lipids comprising quaternary ammonium salts that are advantageously (but not absolutely) suitable for plasmids are advantageously lipids having the formula:

In which R ₁ is a saturated or unsaturated linear aliphatic group having 12 to 18 carbon atoms, R ₂ is another aliphatic group having 2 or 3 carbon atoms, and X is an amine or hydroxyl group (eg DMRIE). In another embodiment, the cationic lipid may be associated with a neutral lipid (eg, DOPE).
Among these cationic lipids, DMRIE (N- (2-hydroxyethyl) -N, N-dimethyl-2,3-bis (tetradecyloxy) -1-propaneammonium; WO96 / 34109) is particularly preferred, DMRIE-DOPE is preferably formed with neutral lipids (preferably DOPE (dioleoyl-phosphatidyl-ethanolamine; Behr, 1994)).
Advantageously, a plasmid mixture comprising an adjuvant is formed instantly, advantageously formed simultaneously with the administration of the preparation or shortly before administration of the preparation, e.g. forming a plasmid-adjuvant mixture shortly before administration, Advantageously, sufficient time is allowed prior to administration to form a complex in the mixture, such as from about 10 minutes to about 60 minutes prior to administration, for example about 30 minutes prior to administration.
When DOPE is present, the DMRIE: DOPE molar ratio is advantageously about 95: about 5 to about 5: about 95, more advantageously about 1: about 1, for example 1: 1.
DMRIE or DMRIE-DOPE adjuvant: plasmid mass ratio is about 50: about 1 to about 1: about 10, for example about 10: about 1 to about 1: about 5, advantageously about 1: about 1 to about 1 : About 2, for example 1: 1 to 1: 2.

  In another embodiment, the pharmaceutically or veterinarily acceptable carrier, excipient, or vehicle can be a water-in-oil emulsion. Suitable water-in-oil emulsions include water-in-oil vaccine emulsions based on oils that are stable and liquid at 4 ° C. Said vaccine emulsion is an antigen-containing aqueous phase of 6 to 50 v / v% (preferably 12 to 25 v / v%), an oil phase of 50 to 94 v / v%, in whole or in part as non-metabolizable oil (eg 0.2 to 20 p / v% (preferably 3 to 8 p / v%) surfactant, including mineral oils such as paraffin oils and / or metabolic oils such as vegetable oils or fatty acids, polyols or alcohol esters And the latter is any polyglycerol ester, in whole or in part or as a mixture, preferably said polyglycerol ester is polyglycerol (poly) lysine oleate or polyoxyethylene lysine oil or other hydrogenated polyoxy Ethylene lysine oil. Examples of surfactants that can be used in water-in-oil emulsions include ethoxylated sorbitan esters (eg, polyoxyethylene (20) sorbitan monooleate (Tween 80 ™); AppliChem, Inc. (Cheshire, CT ) And sorbitan esters (eg, sorbitan monooleate (Span 80 ™; available from Sigma Aldrich (St. Louis, Mo.)). See also US Patent No. 6,919,084 (eg, Example 8 thereof), which is incorporated herein by reference.In some embodiments, the antigen-containing aqueous phase contains one or more than one An example of a suitable buffer solution is phosphate buffered saline.In a preferred embodiment, the water-in-oil emulsion is a water / oil / water triple emulsion. (See, eg, US Pat. No. 6,358,500, which is incorporated herein by reference.) Examples of other suitable emulsions are described in US Pat. No. 7,371,395, which is incorporated herein by reference. include).

The immunological composition or vaccine of the invention comprises or essentially consists of one or more adjuvants. Suitable adjuvants used in the practice of the present invention are: (1) acrylic or methacrylic acid polymers, maleic anhydride and alkenyl derivative polymers, (2) immunostimulatory sequences (ISS), eg one or more Oligodeoxyribonucleotides having unmethylated CpG units (Klinman et al. 1996; WO98 / 16247), (3) oil-in-water emulsions such as SPT emulsions described on page 147 of the following literature (M. Powell, M. Newman, “Vaccine Design, The Subunit and Adjuvant Approach”, Plenum Press 1995) and MF59 emulsion (described on page 183 of the same document), (4) Cationic lipids containing quaternary ammonium salts, eg DDA , (5) cytokines, (6) aluminum hydroxide or phosphate, (7) saponin or (8) by citation and reference to this application Other adjuvants are discussed in Murrell any document, or (9) any combinations or mixtures of the.
Oil-in-water emulsions (3), which are particularly suitable for viral vectors, can be based on: liquid light paraffin oil (European Pharmacopoeia type), isoprenoid oil (eg squalane, squalene), alkene ( Oils resulting from oligomerization of eg isobutene or decene), esters of acids or alcohols with linear alkyl groups, such as vegetable oil, ethyl oleate, propylene glycol, di (caprylate / caprate), glycerol tri (caprylate / caprate) And propylene glycol dioleate, or esters of branched fatty alcohols or acids, especially isostearates.

The oil is used with an emulsifier to form an emulsion. The emulsifier may be a nonionic surfactant, for example: sorbitan, mannide (eg anhydromannitol oleate), glycerol, polyglycerol or propyleneglycerol ester on the one hand, and oleic acid, isostearic acid, ricinol on the other hand Acids, esters of hydroxystearic acid (the esters are optionally ethoxylated), or polyoxypropylene-polyoxyethylene copolymer blocks such as pluronics such as L121.
Among the type (1) adjuvant polymers, polymers of cross-linked (especially cross-linked by sugars or polyalkenyl ethers of polyalcohols) acrylic or methacrylic acid are particularly preferred. These compounds are known by the carbomer name (Pharmeuropa, 8 (2), June 1996). One skilled in the art can also refer to US Pat. No. 2,909,462. Said document provides such acrylic acid polymers crosslinked by polyhydroxyl compounds. Said polyhydroxyl compound preferably has at least 3 hydroxyl groups, not exceeding 8, wherein the hydrogen atoms of at least 3 hydroxyl groups are replaced by unsaturated aliphatic radicals having at least 2 carbon atoms. Preferred radicals are those containing 2 to 4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated groups. Unsaturated radicals can also contain other substituents such as methyl. A product sold under the name Cabopol (BF Goodrich, Ohio, USA) is particularly suitable. The product is crosslinked with allyl saccharose or allyl pentaerythritol. Among those products, mention may be made in particular of Carbopol 974P, 934P and 971P.

For maleic anhydride-alkenyl derivative copolymers, EMA (Monsanto) is preferred. The above are linear or cross-linked ethylene-maleic anhydride copolymers, which are cross-linked, for example by divinyl ether. The following literature is also available (J Fields et al. 1960).
In terms of structure, acrylic or methacrylic acid polymers and EMA are preferably formed by basic units having the following formula:

Where
R1 and R2 (which may be the same or different) represent H or CH ₃
x = 0 or 1, preferably x = 1,
y = 1 or 2 and x + y = 2.
For EMA, x = 0 and y = 2, and for carbomer, x = y = 1.
These polymers are soluble in water or physiological salt solution (20 g / L NaCl) and the pH is adjusted from 7.3 to 7.4, for example with soda (NaOH), to provide an adjuvant solution that can incorporate the expression vector. be able to. The polymer concentration in the final immunological or vaccine composition can range from 0.01 to 1.5% w / v, 0.05 to 1% w / v, or 0.1 to 0.4% w / v.

One or more cytokines (5) may be in the form of a protein in an immunological or vaccine composition, or may be co-expressed in a host with one or more immunogens or epitopes thereof . Co-expression of one or more cytokines with the same vector as that expressing the one or more immunogens or epitopes or with separate vectors is preferred.
The present invention comprises preparing such a combination composition, for example by mixing a plurality of active compounds advantageously together with further adjuvants, carriers, cytokines and / or diluents.
Cytokines that can be used in the present invention include granulocyte colony stimulating factor (G-CSF), granulocyte / macrophage colony stimulating factor (GM-CSF), interferon α (IFNα), interferon β (IFNβ), interferon γ ( IFNγ), interleukin-1α (IL-1α), interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL- 4), Interleukin-5 (IL-5), Interleukin-6 (IL-6), Interleukin-7 (IL-7), Interleukin-8 (IL-8), Interleukin-9 (IL- 9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), tumor necrosis factor α (TNFα), tumor necrosis factor β (TNFβ), and Includes, but is not limited to, transforming growth factor beta (TGFbeta) It will be appreciated that the cytokines can be administered simultaneously with and / or continuously with the immunological or vaccine composition of the invention. Thus, for example, a vaccine of the invention can also include an exogenous nucleic acid molecule that expresses the appropriate cytokine in vivo. Suitable cytokines are, for example, cytokines that are compatible with the host to be immunized or to which an immunological response is to be elicited (eg, a tricytokine for preparations administered to birds).

In another embodiment, the compositions of the present invention can be prepared using chemical or physical methods described in the following literature (Stauffer et al. Recent Patents on Anti-Infective Drug Discovery, 1: 291-296, 2006). Some of the inactivation techniques are summarized in the table below.
In certain embodiments, the vaccine is administered to birds (eg, chicken, duck, cancer, turkey, guinea fowl, partridge or ostrich). Chickens include, but are not limited to, egg-laying chickens, breeding chickens, broilers, pet chickens and cockfights. Ducks include, but are not limited to, Peking duck, musk duck, mule duck and wild duck. In embodiments where the bird is a larger bird than a duck or chicken, larger doses may be contemplated. For example, in embodiments where administration is performed by eye drop, the application dose estimates are 1 chicken dose, 2 chicken dose, 3 chicken dose, 4 chicken dose, 5 chicken dose, 6 chicken dose, 7 chicken dose, 8 chicken dose 9 chicken doses, preferably 10 chicken doses, where necessary, dosages range from about 20 chicken doses to about 100 chicken doses. For reference, the 5.5 log ₁₀ 50% egg infection dose (EID ₅₀ ) is approximately 1 chicken dose.
In another embodiment, the dosage can be the mean embryonic infection dose (EID ₅₀ ). In certain embodiments, the dosage is about 10 ¹ EID ₅₀ , about 10 ² EID ₅₀ , about 10 ³ EID ₅₀ , about 10 ³ EID ₅₀ , about 10 ⁴ EID ₅₀ , about 10 ⁵ EID ₅₀ , about 10 ⁶ EID _50. , About 10 ⁷ EID ₅₀ , or about 10 ⁸ EID ₅₀ .

The immunological compositions and / or vaccines of the present invention comprise or are effective in eliciting a protective or therapeutic response of one or more expression vectors discussed herein. Or consist of the above. Effective amounts can be determined from the present disclosure (including literature incorporated herein) and knowledge in the art without undue experimentation.
Advantageously, when the antigen is hemagglutinin, the dosage is measured in hemagglutinating units (HAU) or micrograms. In advantageous embodiments, the dosage may be about 100 hemagglutinating units (HAU) / dose, about 1000 HAU / dose or about 10000 HAU / dose. In certain embodiments, the dosage is 1 to 100 μg. The dosage volume is about 0.02 mL to 2 mL, preferably 0.03 mL to 1 mL, more preferably 0.03 mL to 0.5 mL, and in a particularly advantageous embodiment, the volume can be about 0.03 mL to about 0.3 mL.
The vaccines of the present invention can be obtained by inoculation (in ovo) via drinking water, spray, aerosol, intranasal instillation, eye drops, beak dipping, wing-web puncture, transdermal, subcutaneous or intramuscular injection. Can be administered. Advantageously, the vaccine is administered by subcutaneous, oocyte inoculation, eye drop, spray or drinking water.
In yet another embodiment, the vaccine is inoculated 1 to 3 days prior to hatching, or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days 8 days old, 9 days old, 10 days old, 11 days old, 12 days old, 13 days old, 14 days old, 15 days old, 16 days old, 17 days old, 18 days old, 19 days old, 20 It can be administered to day-old and 21-day-old chickens.

In certain embodiments of the invention, a prime-boost immunization schedule may be used. It consists of at least one main dose and at least one booster dose (using at least one common protein, polypeptide, antigen, epitope or immunogen). The immunological composition or vaccine used in the main administration is different in nature from that used as a booster. However, it should be noted that the same composition can be used as the main dose and boost. This dosing protocol is referred to as “prime-boost”.
In another aspect of the prime-boost protocol of the present invention, a recombinant viral vector comprising avian influenza antigen and expressing it in vivo after administration of a composition comprising an engineered avian influenza Avinew NDV vaccine or composition A vaccine or composition comprising, or an inactivated virus vaccine or composition comprising an avian influenza antigen, or a vaccine or composition comprising an avian influenza subunit (protein), or a DNA plasmid vaccine comprising and expressing an avian influenza antigen or The composition is administered. Similarly, a prime-boost protocol may include a vaccine or composition comprising a recombinant viral vector that contains and expresses an avian influenza antigen in vivo, or an inactivated virus vaccine or composition comprising an avian influenza antigen, or an avian influenza sub- Administration of a composition comprising a united avian influenza Avinew NDV vaccine or composition after administration of a vaccine or composition comprising a unit (protein) or a DNA plasmid vaccine or composition comprising and expressing an avian influenza antigen it can. Furthermore, it should be noted that both primary and secondary administration can include a composition comprising an engineered avian influenza Avinew NDV vaccine or composition.

The prime-boost protocol includes at least one prime dose and at least one boost dose with at least one common antigen. The vaccine or composition used for prime administration may be different in nature from that used as a subsequent booster vaccine or composition. Prime administration can include one or more administrations. Similarly, boost administration can include one or more administrations.
Preferably, the various administrations are performed about 1 to 6 weeks apart, or about 2 to 4 weeks apart. A booster that repeats every 2 to 6 weeks or every year is also contemplated. Preferably, the animal is at least 1 day old at the first administration.
The immunological composition and / or vaccine comprises about 10 ⁴ to about 10 ¹¹ , preferably about 10 ⁵ to about 10 ¹⁰ , more preferably about 10 ⁶ to about 10 ⁹ influenza antigens, epitopes per dose. Or a virus particle of a recombinant adenovirus that expresses the immunogen. In the case of immunological compositions and / or vaccines based on poxvirus, one dose is 10 ² pfu to 10 ⁹ pfu. The immunological composition and / or vaccine is a poxvirus or herpesvirus expressing an influenza antigen, epitope or immunogen of from about 10 ² to about 10 ⁷ , preferably from about 10 ³ to about 10 ⁵ pfu per dose. Includes recombinants.
In embodiments where the avian immunological composition or vaccine is an inactivated avian virus, the inactivated avian virus can be derived from various species viruses used in the manufacture of oil emulsion vaccines, Ulster 2C, B1, LaSota Or Roakin may be included. The inactivated avian influenza virus may also be a classical inactivated virus (total virus beta-propiolactone (BPL including H5N9 Eurasian isolate A / chicken / Italy 22A / 98 grown in developing SPF eggs) ) Inactivated vaccine (H5N9-It)). Other inactivated vaccines (adjuvanted) include: all commercially available viral preparations based on H5N2 and H5N9 subtype wild viruses (Fort Dodge Animal Health, Intervet International, Merial Italia), or H5N3 induced by genetic engineering (the latter are the modified HA gene of A / chicken / Vietnam / C58 / 04 (H5N1), the neuraminidase gene of A / duck / Germany / 1215/73 (H2N3), and A / PR / 8 / 34 (including the internal gene of H1N1)).

The viral vector can be an attenuated avipox expression vector. In certain embodiments, the avipox expression vector can be a fowlpox vector, such as TROVAC ™. In another embodiment, the avipox expression vector can be a canalipox vector, such as ALVAC ™. The influenza antigen, epitope or immunogen can be a hemagglutinin, such as H5. The fowlpox vector can be vFP89 (see US2008 / 0107681 and US2008 / 0107687) or vFP2211 (see US2008 / 0107681 and US2008 / 0107687). The canalipox vector can be vCP2241 (see US2008 / 0107681 and US2008 / 0107687). Other viruses that can be used in the methods of the invention include (but are not limited to): vaccinia viruses such as attenuated vaccinia viruses such as NYVAC, adenoviruses and herpes viruses.
The effectiveness of the vaccine can be tested by challenging animals (eg, birds) with influenza pathogenic strains (eg, influenza H5N1, H5N8 or H5N9 strains) about 2 to 4 weeks after the last immunization. Both homologous and heterologous strains can be used in vaccine efficacy testing challenges. Animals can be challenged by nebulization, intranasal, instillation, ocular nose, IM, intratracheal, and / or oral. The challenge virus can be a total of 10 ³ to 10 ⁸ EID ₅₀ depending on the route of administration. For example, if administration is by nebulization, the virus suspension is aerosolized to produce about 1 to 100 μm droplets, and if administration is intranasal, intratracheal or oral, the total challenge virus is about 0.05 to about 5 mL. The dose volume of the target species specific composition, for example, the dose volume of the avian composition is about 50 μL for egg inoculation, about 20 to about 50 μL for eye drops, and about 0.25 mL to about 1 mL for nebulization. Animals can be observed for clinical symptoms and mortality for 14 days daily after challenge. Furthermore, animal groups can be euthanized and pathological findings can be evaluated. Oropharynx, trachea or total excretory swabs can be collected for virus detection from all animals after challenge. The presence or absence of a viral antigen in a tissue can be determined by immunohistochemistry, virus isolation or titration, or nucleic acid detection (eg, reverse transcriptase polymerase chain reaction (RT-PCR)). Blood samples can be collected after challenge and analyzed for the presence of anti-influenza H5N1 virus specific antibodies.

It will be appreciated by persons skilled in the art that the disclosure herein is provided by way of example and that the present invention is not limited thereto. From the disclosure herein and information in the art, one of ordinary skill in the art can determine the number of doses, route of administration, and dose to be used for each immunization protocol without undue experimentation.
The present invention contemplates administering an effective amount of a therapeutic composition made in accordance with the present invention to an animal at least once. This administration can be via a variety of routes including, but not limited to, intramuscular (IM), intradermal (ID) or subcutaneous (SC) injection, or intranasal, egg or oral administration. The therapeutic compositions of the present invention can also be administered by a needleless device (eg, Pigjet, Dermojet, Biojector, Vetjet or Vitajet device (Biojet, Oregon, USA)). In certain embodiments, the animal is a bird.
Another embodiment of the invention is a kit for performing a method of inducing an immunological or protective response against influenza in an animal. The kit includes instructions for performing a recombinant NDV immunological composition or vaccine or inactivated influenza immunological composition or vaccine, and a method of delivery in an amount effective to elicit an immune response in an animal. including.

Some specific embodiments of the present invention are listed as examples.
(1) A composition comprising an engineered NDV vector.
(2) The composition according to (1), wherein the engineered NDV vector comprises a heterologous polynucleotide encoding an avian influenza HA antigen or a variant thereof.
(3) the engineered NDV vector is at least 90% of a polynucleotide having a sequence identity of at least 90% with the polynucleotide having the sequence shown in SEQ ID NO: 1 or the polynucleotide having the sequence shown in SEQ ID NO: 1 The composition according to (1) or (2), comprising a polynucleotide complementary to a polynucleotide having the sequence identity of:
(4) The composition according to (2) or (3), wherein the avian influenza HA antigen is an H5 HA polypeptide.
(5) The composition according to any one of (2) to (4), wherein the HA antigen has a modified cleavage site.
(6) The HA antigen has at least 80% amino acid sequence identity with a polypeptide having the sequence shown in SEQ ID NO: 15, 17, 19, 21, or 23, according to any one of (2) to (5) Composition.
(7) The heterologous polynucleotide has at least 80% sequence identity with a polynucleotide having the sequence shown in SEQ ID NO: 14, 16, 18, 20, or 22, according to any one of (2) to (6) Composition.
(8) A method for producing an engineered NDV vector by modifying the genome of NDV, the method comprising the step of introducing an isolated polynucleotide into the non-essential region of the NDV genome.
(9) A polynucleotide in which NDV has at least 90% sequence identity with the polynucleotide having the sequence shown in SEQ ID NO: 1, or at least 90% sequence identity with the polynucleotide having the sequence shown in SEQ ID NO: 1. The method according to (8), comprising a polynucleotide that is complementary to the polynucleotide having the polynucleotide.
(10) The non-essential region is selected from a region consisting of an untranslated region located upstream of the NP gene, between two genes of the APMV genome, and downstream of the L gene, (8) or (9) Method.
(11) The method according to any one of (8) to (10), wherein the non-essential region is located between the P and M genes or between the M and F genes.
(12) A method of inducing or immunizing a bird against at least one avian pathogen, comprising administering an engineered NDV vector that expresses at least one antigen.
(13) A polynucleotide in which the NDV vector has at least 90% sequence identity with the polynucleotide having the sequence shown in SEQ ID NO: 1, or at least 90% sequence identity with the polynucleotide having the sequence shown in SEQ ID NO: 1 The method according to (12), comprising a polynucleotide complementary to a polynucleotide having
(14) The method according to (12) or (13), wherein the antigen is an avian influenza antigen.
(15) The method according to (14), wherein the avian influenza antigen is hemagglutinin (HA).
(16) The method according to (15), wherein the avian HA antigen has at least 80% sequence identity with a polypeptide having the sequence shown in SEQ ID NO: 15, 17, 19, 21, or 23.
(17) The method according to any one of (12) to (16), wherein administration is by eye drop, spray, drinking water, egg inoculation, intramuscular or subcutaneous administration.
(18) The method according to any one of (12) to (17), wherein the administration is prime-boost.
(19) A vaccine or composition comprising a prime-boost wherein the composition according to any one of (1) to (7) is prime-administered and a recombinant viral vector comprising an avian pathogen antigen and expressing it in vivo Or an inactivated virus vaccine comprising an avian pathogen antigen, or a vaccine or composition comprising an avian pathogen antigen (subunit), or a boost-administration of a DNA plasmid vaccine or composition comprising and expressing an avian pathogen antigen (18) The method as described in (18).
(20) A vaccine or composition comprising a recombinant viral vector in which prime-boost contains and expresses an avian pathogen antigen in vivo, or an inactivated virus vaccine comprising an avian pathogen antigen, or an avian pathogen antigen (subunit) Or a DNA plasmid vaccine or composition containing and expressing an avian pathogen antigen and boost-administration of a composition according to any of (1) to (7) (18) The method as described in (18).
(21) The prime-administration comprises a composition comprising a poxvirus that contains and expresses an avian influenza antigen, and the boost-administration further comprises an avian influenza antigen. The method according to (20), comprising the composition of:
(22) Prime-boost comprises prime-administration of the composition according to any of (1) to (7) and boost-administration of the composition according to any of (1) to (7), The method according to 18).
(23) The method according to any one of (12) to (22), wherein the bird is a chicken or a duck.
(24) An engineered NDV vector comprising a polynucleotide having at least 90% sequence identity to SEQ ID NO: 1.
(25) A polynucleotide selected from the group consisting of:
a) a polynucleotide having at least 90% sequence identity to a polynucleotide having the sequence shown in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12 or 24;
b) a polynucleotide having at least 90% sequence identity with a polynucleotide encoding a polypeptide having the sequence shown in SEQ ID NO: 3, 5, 7, 9, 11 or 13;
c) A polynucleotide complementary to the polynucleotide described in a) or b).

The invention is further illustrated by the following non-limiting examples.
Example
DNA inserts, construction of plasmids and recombinant viral vectors, J Sambrook et al were performed using standard molecular biology techniques described (Molecular Cloning:. A Laboratory Manual , 2 nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989).

Development of reverse genetics of AVINEW (TM) (NDV) strain and production of NDV mutant expressing heterologous gene The purpose of this example is to develop reverse genetics of AVINEW NDV strain and manipulate heterogeneous gene expressing NDV mutant Was to make.
NDV is a minus RNA virus containing 6 major genes (NP, P, M, F, HN and L) as shown in FIG. 1A. The production of genetically modified NDV viruses requires a reverse genetics system. Applicants have developed a reverse genetics system based on NDV's AVINEW vaccine strain. This system allows for the generation of modified Newcastle disease viruses that express foreign genes, such as influenza hemagglutinin (HA), as shown in FIG. 1B.
Example 1.1: Cloning and sequencing of the entire AVINEW NDV genome on a transcription plasmid
For the purpose of sequencing the genome of the AVINEW NDV strain, it is necessary to clone the entire genome of the AVINEW strain with a plasmid called “transcript plasmid” (see FIG. 1C). The transcribed plasmid allows for the generation of positive RNA that matches the complete genome of the AVINEW strain of NDV. A method for cloning the AVINEW genome by sequentially joining a set of 10 overlapping cDNA fragments amplified from AVINEW-extracted RNA by reverse transcriptase polymerase chain reaction (RT-PCR) is shown in Figure 2. . This final plasmid, called pIV029 (see Figure 3), contains the complete genomic sequence of AVINEW under the control of the T7 RNA polymerase transcription signal (the upstream T7 promoter), and hepatitis delta virus (HDV) Terminate with a ribozyme (the ribozyme is used to cleave RNA at the true NDV genome termination point followed by a T7 terminator). A restriction site is inserted between P and M to allow insertion of the transgene (see Figures 2 and 3).
The complete genome sequence of the AVINEW ™ strain was determined. The AVINEW ™ genome is 15186 bp in length, which is a multiple of 6 nucleotides as expected based on the nucleocapsid protein binding motif.
The commented sequence of the insert of pIV029 is presented in FIGS. 4A-4O and the plasmid map is presented in FIG. In FIGS. 4A-4O, the six open reading frames (ORFs) (NP, P, M, F, HN and L) of the Avinew strain are translated into their amino acid sequences. Each ORF is flanked by a “gene start” upstream sequence and a “gene end” downstream sequence indicated by GS and GE. T7 promoter and T7 terminator sequences are displayed.

Example 1.2: Construction of an expression plasmid containing the NP, P and L genes of AVINEW NDV In the reverse genetics system, the nucleocapsid (NP), phosphoprotein (P) and large polymerase protein (L) under the control of the T7 RNA polymerase promoter. It is necessary to construct a plasmid called “expression plasmid” encoding (see FIG. 1C). These three proteins bind to viral RNA to form the smallest NDV infection unit, ribonucleoprotein (RNP). This complex comprising NP, P and L proteins exhibits RNA-dependent RNA polymerase activity.
Expression plasmids pIV32 (Figure 5), pIV33 (Figure 6) and pIV34 (Figure 7) were constructed and under control of the T7 RNA polymerase promoter and the foot-and-mouth disease virus (FMDV) internal ribosome entry site (IRES), respectively NP, P And contains the L gene.
Example 1.2.1: Construction of an expression plasmid pIV32 containing the NP gene of AVINEW NDV A map of the expression plasmid pIV32 is shown in FIG. This plasmid is the open reading frame (ORF) of the nucleocapsid (NP) gene of the Newcastle disease virus AVINEW ™ vaccine strain under the control of the T7 RNA polymerase promoter and the internal ribosome entry site (IRES) of foot-and-mouth disease virus (FMDV). The nucleotide sequence encoding was included. NDV ORF NP (1467 bp, SEQ ID NO: 2) encodes a 489 amino acid polypeptide (SEQ ID NO: 3). Protein NP is the main structural component of nucleocapsid. This protein is approximately 58 kDa. A total of 2600 NP molecules tightly wrap the genomic RNA. NP interacts with several other virally encoded proteins, all of which are required for replication control. (NP-NP, NP-P, NP- (PL) and NP-V). NP combined with NDV genomic RNA and proteins P and L constitutes an NDV ribonucleoprotein (RNP) complex, which is an infectious form of the NVD genome.

Example 1.2.2: Construction of an expression plasmid pIV33 containing the P gene of AVINEW NDV A map of the expression plasmid pIV33 is shown in FIG. This plasmid is a nucleotide sequence encoding the ORF of the phosphoprotein (P) gene of the Newcastle disease virus AVINEW ™ vaccine strain under the control of the T7 RNA polymerase promoter and the internal ribosome entry site (IRES) of foot-and-mouth disease virus (FMDV) Was included. This plasmid was designed to create recombinant NDV AVINEW ™ as a vaccine vector using reverse genetics methodology.
NDV ORF P (1185 bp, SEQ ID NO: 4) encodes a 395 amino acid polypeptide, namely the structural phosphoprotein (SEQ ID NO: 5). This protein has a molecular weight of 53 to 56 kDa as determined by SDS-PAGE. Protein P is essential for the activity of the RNA polymerase complex, which forms the complex with the large subunit L. Although the catalytic activity of the polymerase is bound to the L subunit, its function requires specific interaction with P. Proteins L and NP and P bound to NDV genomic RNA constitute an NDV ribonucleoprotein (RNP) complex, which is an infectious form of the NVD genome.
Furthermore, the P gene encodes protein V (function unknown) with an apparent molecular weight of 36 to 38 kDa on SDS-PAGE. P and V proteins share the same amino terminus, but their C-terminus is diverse. This difference is caused by the RNA editing mechanism, in which one G residue that is not templated is inserted into the P gene-derived mRNA. The unedited transcript encodes the P protein, while the edited transcript encodes the V protein. Because they are phosphoproteins, P and V are rich in serine and threonine residues over their entire length. Furthermore, the V protein is rich in cysteine residues at the C-terminus. Similarly, the P gene encodes protein W (function unknown) with an apparent molecular weight of 28 to 33 kDa on SDS-PAGE. This protein is also generated by an RNA editing mechanism, in which two are inserted into the P gene-derived mRNA instead of one template-independent G residue.

Example 1.2.3: Construction of an expression plasmid pIV34 containing the L gene of AVINEW NDV A map of the expression plasmid pIV34 is shown in FIG. This plasmid is a nucleotide encoding the ORF of the large polymerase protein (L) gene of the Newcastle disease virus AVINEW ™ vaccine strain under the control of the T7 RNA polymerase promoter and the internal ribosome entry site (IRES) of foot-and-mouth disease virus (FMDV) Contained sequences. This plasmid was designed to create recombinant NDV AVINEW ™ as a vaccine vector using reverse genetics methodology.
NDV L (6612 bp, SEQ ID NO: 12) encodes a 2204 amino acid polypeptide, which is an L protein (SEQ ID NO: 13). The Paramyxoviridae, like other non-segmented negative-strand RNA viruses, has an RNA-dependent RNA polymerase composed of two subunits (large protein L and phosphoprotein P). The L protein imparts RNA polymerase activity to the complex, while the P protein functions as a transcription factor. Protein P and NP and protein L combined with NDV genomic RNA constitute an NDV ribonucleoprotein (RNP) complex, which is an infectious form of the NVD genome.
Example 1.3: Construction of an expression plasmid that expresses T7 RNA polymerase The reverse genetics system requires that T7 RNA polymerase be expressed in the cell, in which case the NDV virus will be regenerated (see FIG. 1C). . A variety of systems can be used to express T7 RNA polymerase. These include the use of recombinant viruses (eg, avipox) as vectors, the use of cells that constitutively express the enzyme, or transient expression using expression plasmids. The latter solution was chosen and an expression plasmid (called pNS151) encoding T7 RNA polymerase under the HCMV IE promoter was constructed. T7 RNA polymerase not only allows transcription / expression of NP, P and L proteins from the expression plasmids described above, but also transcribes the NDV genome (present in the transcription plasmid) into a positive-sense RNA. A map of this plasmid is shown in FIG.

Example 1.4: Recovery of NDV virus using reverse genetics system Five plasmids described above (one is a transcription plasmid containing the NDV genome, three are expression plasmids expressing NDV NP, P and L, and T7 An expression plasmid expressing the polymerase) was cotransfected together into Chinese hamster ovary (CHO) cells. FIG. 1C is a schematic diagram for explaining the mechanism of the reverse genetics system. The schematic shows that upon entry into the cell, T7 RNA polymerase is expressed, which then transcribes the NDV genome from the transcription plasmid into the plus-sense RNA (RNA (+)) genome, as well as NP, P and L Shows that genes are transcribed from individual expression plasmids. Subsequently, the NP, P and L protein transcripts are translated as expressed NP, P and L proteins, which are subsequently assembled to form RNPs with genomic RNA (+). This RNP complex synthesizes a negative-strand RNA genome (RNA (-)), which subsequently initiates the normal replication cycle of the NDV virus and promotes the generation of infectious particles. Trypsin or other exogenous proteases such as provided by egg urinary sac can be added to the culture to cleave the viral F protein produced.
Using this system, infectious particles of AVINEW NDV can be obtained. In short, various required plasmids (pIV029, pIV32, pIV33, pIV034 and pNS151) were transfected into CHO cells. After 72 hours, the CHO supernatant was inoculated into 10-day-old embryonated eggs to amplify the virus. Three days later, urinary sac fluid was collected and hemagglutination activity (HA) was checked using chicken erythrocytes. The resulting reverse genetic AVINEW mutant was designated vAVW01. It contained the same sequence as the AVINEW parent virus except for two unique restriction sites (PacI and FseI) introduced between the P and M genes (see FIG. 2).

Example 1.5: Production of NDV virus expressing foreign gene using reverse genetic system In order to produce a modified NDV virus expressing a foreign antigen, selection of the insertion locus was necessary. Various loci can be selected, including upstream of the NP gene and between the two genes. In this example, a site between the P and M genes of AVINEW ™ NDV was selected and a foreign gene was inserted as shown in FIG. The foreign gene must be inserted with the necessary “start” and “stop” transcription sequences, and the number of inserted nucleotides should be scheduled so that the total number of nucleotides in the modified NDV genome remains a multiple of 6. There is.
This example details the generation of an AVINEW mutant that expresses the avian influenza-derived hemagglutinin (HA) gene. The HA gene was first inserted into a transfer plasmid (this plasmid allows the insertion of foreign genes and flanking sequences into the PacI and FseI specific restriction sites of pIV029). The structure of the transfer plasmid is shown in FIG. 9 (in frame). From left to right, PacI site, P3IUTR downstream from PacI site, P gene end point (or STOP) sequence, P / M intergene, M gene start (or START) sequence, M It contains a 5 ′ UTR, a multiple cloning site, a P ′ (upstream from the PacI site) 3′UTR and FseI sites. The HA gene was cloned at the multiple cloning site of the transfer plasmid described in FIG. 9, followed by cloning of the entire PacI / FseI insert at the same restriction site of pIV029 to generate a transcription plasmid containing the foreign gene. An example of such a transcription plasmid (pIV039) is shown in FIG. In this example, a synthetic gene modified at the cleavage site encoding the highly pathogenic H5N1 avian influenza virus A / chicken / Indonesia / 7/2003 HA gene amino acid sequence was inserted into the transcription plasmid.
Highly pathogenic (HP) H5N1 avian influenza using the transcription plasmid pIV039 together with four additional expression plasmids (see Example 1.4) required for the generation of AVINEW mutants by reverse genetics (AI) An AVINEW ™ mutant called vAVW02 that expresses the HA gene from virus A / chicken / Indonesia / 7/2003 was made.
Using the same method, various AVINEW mutants expressing HA genes from various HPAI H5N1 or low pathogenic AI (LPAI) H9N2 were generated. The sequence of the inserted HA gene from various H5N1 and H9N2 AI isolates has been assigned the SEQ ID NO shown in FIG. 12, and both DNA and protein sequences are included in the sequence listing. The content of the sequence listing filed electronically with the application documents is hereby incorporated by reference in its entirety.
The method described above was successfully used for the recovery of infectious AVINEW mutants expressing HA genes from various HPAI H5N1 or low pathogenic AI (LPAI) H9N2 (see Table 1).

Table 1: Various Avinew mutants made from various avian influenza isolates and expressing a synthetic HA gene from said isolate. To match the sequence of the cleavage site of the LPAI H5 isolate, mutations were introduced into the cleavage sites of all HA genes from HPAI H5N1.

Example 1.6: Production and characterization of NDV virus expressing foreign gene using reverse genetics system All amplified AVINEW mutants were subcultured in the developing egg to determine the characterization. Recombinant virus grows to a titer similar to the original AVINEW ™ virus (8 to 10 log EID ₅₀ / mL), and foreign gene insertion may not significantly affect virus replication in the developing egg It was suggested. Examples of infectious titers obtained in the second or third round of developmental eggs are shown in Table 2.
H5 transgene expression was confirmed by indirect immunofluorescence in infected CHO cells and immunoblot (Western blot or WB) in urinary sac and CHO infected cell lysates (see, eg, FIGS. 11A and 11B for vAVW02) ). The electrophoresis profile of H5 was as expected. Due to the presence of proteases in the urinary sac, appropriate HA cleavage products (ie HA1 (50 kDa) and HA2 (28 kDa) were detected. In CHO cells infected with egg-grown virus (in the absence of trypsin) ), Applicants detected only HA0 type (75 kDa) (FIG. 11A).
The expression of foreign HA protein on the surface of NDV virion was confirmed by immunoelectron microscopy using vAVW02 as an example (see FIG. 2B).

Table 2: Yield and expression control of Avinew mutants produced from various avian influenza isolates and expressing the synthetic HA gene derived from the above

Chicken Experiment 1: Protection against Newcastle disease induced in chicken by engineered AVINEW mutant and ND and AI HI titers The purpose of this experiment is to protect chicken against Newcastle disease (ND) by inserting foreign genes into the genome of AVINEW strain It was to prove that the ability was not reduced. The vaccine immunization schedule is shown in the upper panel of FIG. 3A. The protection rates induced by the AVINEW vaccine and the two engineered AVINEW mutants (vAVW01 without any insert (see Table 1) and vAVW03 with the HPAI H5N1 HA insert) are shown in the bottom table of Figure 3A . Similar levels of ND protection were induced by three test doses of the three vaccines, indicating that under these test conditions there was no negative effect of HA insertion on the vector's ability to protect against velogenic NDV challenge by the Herts33 strain. It was.

Protection against H5N1 HPAI induced by engineered AVINEW mutants in SPF chickens with or without maternally derived antibody (MDA) against NDV and / or AI
Example 3.1: Chicken Experiment 2: Protected against Hungary (2006) H5N1 HPAI induced in SPF chickens by a single administration of engineered AVINEW mutant SP no, it was evaluated by the s pecific p athogen f ree) chicken. Eight 1-day-old SPF chickens were vaccinated with 10 ⁵ EID ₅₀ by the eye drop (ED) and intranasal (IN) routes (D0). H5N1 challenge (6 log10 of H5N1 clade 2.2 A / duck / Hungary / 11804/2006 isolate per chick) was performed 4 weeks (D28) and 6 weeks (D42) after vaccine immunization. Chickens were monitored for up to 2 weeks after challenge. The results are shown in Table 3 and Table A.

Table 3: Chicken experiment 2: Clinical protection results of a vaccine immunization-challenge experiment evaluating the effectiveness of protection against HPAI H5N1 (A / duck / Hungary / 11804/2006) challenge induced in SPF chickens by engineered vAVW03 mutants
^a MTD: Average time to death in days

  Table A: Chicken Experiment 2: Vaccine immunity-challenge experiment virus efflux protection results assessing protection efficacy against HPAI H5N1 (A / duck / Hungary / 11804/2006) challenge induced in engineered vAVW03 mutants in SPF chickens

^a discharge was determined from the mouth and cloaca swabs taken after challenge 2, 4 and 7 days (dpc) using real-time PCR.

  Full and partial (75%) clinical protection was induced with D28 and D42, respectively. The number of chickens excreting detectable amounts of challenge virus decreased in the vaccine immunization group. Furthermore, the viral shedding level at 2 dpc of the vaccine immunization group (2 and 4) was 3 log10 and about 2 log10 or more lower than the control shedding level after challenge with D28 and D42, respectively. Taken together, these results indicate that the Avinew vector expressing the HPAI HA gene is protective in SPF chickens.

Example 3. A: Chicken Experiment A: Protection against Egypt (2006) H5N1 HPAI induced in SPF chickens by a single administration of engineered AVINEW mutant SPF (specificity of AVINEW mutant vAVW03 against Egypt HPAI H5N1 challenge) Evaluated in chickens (no pathogen). Ten 1-day-old SPF chickens were vaccinated with 10 ⁶ EID ₅₀ by the eye drop (ED) and intranasal (IN) routes (D0). H5N1 challenge (6 log10 of H5N1 clade 2.2 A / chicken / Egypt / 06959-NLQP / 2006 isolate per chick) was performed 3 weeks after vaccine immunization (D21). Chickens were monitored for 2 weeks after challenge. The results are shown in Table B.

Table B: Chicken Experiment 2: Clinical Protection Results of Vaccine Immunization-Challenge Experiment to Evaluate Protection Efficacy against HPAI H5N1 (A / Duck / Hungary / 11804/2006) Challenge Induced in SPF Chickens with Engineered vAVW03 Mutants
Excellent (90%) clinical protection was induced against this Egyptian HPAI H5N1 isolate at D21, confirming that the Avinew vector expressing the HPAI HA gene induces protection in SPF chickens.
Example 3.B: Chicken experiment B: One or two doses of engineered AVINEW variant or Hungary (2006) protection against H5N1 HPAI induced in SPF chickens by prime-boost immunization schedule Hungary (2006) ) Efficacy against HPAI H5N1 challenge was evaluated in SPF (no specific pathogen) chickens after one or two doses with inactivated vaccine or prime-boost immunization schedule. Eight 1-day-old SPF chickens were vaccinated against D0 (Groups 2, 3 and 4) and D14 (Group 3) with 10 ⁵ EID ₅₀ by instillation (ED) and intranasal (IN) routes. Group 3 chickens received an inactivated vaccine made with the H5N9 A / chicken / Italy / A22 / 1998 LPAI isolate (0.5 mL / chicken). H5N1 challenge (6 log10 of H5N1 clade 2.2 A / duck / Hungary / 11804/2006 isolate per chicken) was performed 4 weeks after the second vaccination (D42). Chickens were monitored for 2 weeks after challenge. The results are shown in Table C.

Table C: Chicken Experiment B: Protection Results of Vaccine Immunization-Challenge Experiment to Evaluate Protection Effectiveness against HPAI H5N1 (A / Duck / Hungary / 11804/2006) Challenge Induced by Various Vaccine Immunization Schedules Containing vAVW03 Mutants
* MTD: Average time to death in days
** dpc: days after challenge
*** Inactivated (or killed) AI H5N9 vaccine was prepared using H5N9 A / chicken / Italy / A22 / 1998 LPAI isolates.

  Excellent clinical protection was induced in three vaccine immunization groups. Vaccine immunization induced a reduction in the number of chickens that shed virus as well as a reduction in the level of challenge virus shed. The highest defense performance was obtained in Group 4 chickens that received the prime-boost immunization schedule. These results confirm that the Avinew vector expressing the HPAI HA gene induces protection in SPF chickens, and further shows that the level of protection can be improved by a prime-boost immunization schedule.

Example 3. C: Chicken experiment C: Protection against varieties Egyptian (2008) H5N1 HPAI isolates in SPF chickens induced by various vaccine immunization schedules containing engineered vAVW03 variants Various vaccine immunization schedules (see Table D) ) The AVINEW mutant vAVW03 contained in the 2008 variant Egyptian isolate (A / chicken / Egypt / 1709-6 / 2008) is effective against HPAI H5N1 challenge with 1-day-old SPF (specific pathogens) No) evaluated with chickens. The vaccine schedule is shown in Table D. The vAVW03 vaccine 10 ⁶ EID ₅₀ / intraocularly in 100μL dose (50 [mu] L) and intranasal (50 [mu] L) route was administered to D0 (groups 2 and 3) and D14 (group 2 and 4). Group 3 chickens received an inactivated vaccine made using H5N1 clade 2.3 A / duck / Anhui / 2006 LPAI isolate-derived HA (modified at the cleavage site) and reverse genetics H5N1 strain containing NA ( 0.5mL / chicken). Group 4 chickens include fowlpox recombinant vFP89 expressing HA from H5N8 HPAI A / turkey / Ireland / 1378/1983 isolate (see TROVAC-AIV H5 vaccine, US2008 / 0107681 and US2008 / 0107687) was administered to D0 in the neck muscles by subcutaneous route is desired) Marek's disease vaccine diluent (commercially available dose diluted with patent substance) Merial (about 3.5 log10 TCID ₅₀ / 200μL). H5N1 challenge (5 log10 of H5N1 clade 2.2 A / chicken / Egypt / 1709-6 / 2008 isolate per chick) was performed 2 weeks after the second vaccine immunization (D28). Chickens were monitored for 10 days after challenge. The results are shown in Table D.

Table D: Chicken Experiment C: Vaccine immunization assessing protective efficacy in SPF chickens against HPAI H5N1 (A / chicken / Egypt / 1709-6 / 2008) challenge induced by various vaccine immunization schedules containing vAVW03 mutants -Clinical defense results of challenge experiments
* H5N1 antigen was prepared from A / turkey / Turkey / 1/2005 antigen; SD = standard deviation
** MTD: average time to death in days

  The HI titers induced by the various vaccine immunization schedules are shown in Table D. As expected, high HI titers were obtained in group 3 after boosting with the inactivated vaccine. Excellent clinical protection (93-100%) was induced in the three vaccine immunization groups. The A / chicken / Egypt / 1709-6 / 2008 isolate (HA protein sequence available from GenBank (ACD65000.1)) is one of the H5N1 antigen variants that recently appeared in Egypt, It would be worth mentioning that inactivated H5 vaccines can provide lower protection than against older Egyptian strains. These results confirmed that the Avinew vector expressing the HPAI HA gene induces protection against the antigenic variant H5N1 Egypt isolate in SPF chickens.

Example 3.2: Chicken experiment 3: Protection against H5N1 HPAI induced by engineered AVINEW mutant in chickens with and without NDV and / or AI MDA To assess the level of HPAI H5N1 protection induced by vAVW03 in SPF chickens and further assess the interference that can be generated by maternally derived antibodies (MDA) against NDV vectors and / or AI.
In order to evaluate the effect of MDA on AI defense, it was necessary to immunize SPF breeders with various vaccine immunization schedules as shown in Table 4. There were three groups of breeders: The first group (G1) was NDV (three doses of AVINEW and one dose of inactivated (or killed) vaccine) and AI (H5N9 A / chicken) / Italy / A22 / 1998 vaccinated against inactivated vaccine based on LPAI isolate (3 times); second group (G4) vaccinated against NDV only (group 1 and The same immunization schedule); the third group (G5) was vaccinated with AI alone (inactivated vaccine based on H5N1 mutants containing HA and NA genes from A / Gun / Guandon / 1996 isolate) (See Table 4 for details).

Table 4: Vaccination immunization schedule of SPF breeders used to obtain 1-day-old chickens with ND and / or AI MDA
* L = live NDV vaccine AVINEW; K = killed NDV vaccine (Gallimune 407)
** Inactivated (or killed) AI H5N9 vaccine was prepared using H5N9 A / chicken / Italy / A22 / 1998 LPAI isolate.
*** Inactivated (or killed) AI H5N1 vaccine was prepared using AI variants containing HA and NA genes from H5N1 A / Gun / Guandong / 1996 HPAI isolates.

Chickens hatched from eggs laid by these immunized chickens and HPAI H5N1 efficacy induced by vAVW03 in these chickens with MDA was compared to that induced in SPF chickens without MDA.
FIG. 13 shows the mean MDV titer and AI HI titer (log2) observed in 1-day-old chickens hatched from the immune breeders shown in Table 4. NDV titers were very high in both groups of ND vaccine immunized breeders (G1 and G4). AI HI titers measured with the H5N1 clade 2.2 (A / duck / Hungary / 11804/2006) and H5N9 (A / chicken / Italy / A22 / 1998) antigens were derived from chicken offspring of G5 breeders immunized with inactivated H5N1 vaccine It was higher. These results confirmed the presence of the expected MDA in 1-day-old chickens hatched from vaccine-immunized SPF chickens.
FIG. 14 shows the time course of the immunization and challenge protocol for SPF chickens with or without MDA. 1 day old chicken from normal SPF chicken or vaccine immunized SPF breeder chicken, vAVW03 (10 chickens) expressing HA gene from A / turkey / turkey 1/2005 HPAI H5N1 isolate or any HA insert In addition, immunization was carried out by ocular nasal route with 10 ⁵ EID ₅₀ of vAVW01 (10 chickens) used as a control. Three weeks after vaccination, all chickens were challenged by the ocular nasal route with 6 log10 of the HPAI H5N1 A / duck / Hungary / 11804/2006 isolate. Two weeks after challenge, chickens were observed for clinical symptoms and mortality. Table 5 is a summary of vaccine immunization schedule and avian influenza protection results.

Table 5: IA protection induced by H5N1 HA-expressing AVINEW ™ variant (vAVW03) in SPF breeders and chickens with various MDA

  Rapid mortality (mean mortality 3.6-3.8 days) of chickens (groups 1 and 5) without AIVDA vaccinated with vAVW01 proved the challenge (see Table 5 and Figure 15). After a single mucosal administration of vAVW03 to 1 day old SPF chickens, HPAI H5N1 protection level protected 90% of chickens. Surprisingly, all vAVW03 immunized chickens (Group 6) hatched from breeders immunized with NDV alone (G4 in Table 4) are protected from HPAI challenge and there is no anti-vector NDV MDA interference in AI protection It was shown that. Assessment of the impact of AIV MDA was more difficult. Because only 7 out of 10 and 1 died in the control group after challenge (Groups 3 and 7). However, it was shown that 7/9 birds vaccinated with vAVW03 (Group 4) were protected and protection could be induced in the presence of both NDV MAI and AI MDA. Vaccinated chickens that died after challenge died at a later time compared to non-vaccine immunized chickens (see average mortality time in Table 5 and mortality kinetics in Figure 15). Table 6 shows the number of chickens with positive oral and total excretory virus excretion, and FIG. 16 shows the viral excretion kinetics of oral (16A) and total excretory (16B) after challenge. The virus excretion level ratio of vAVW01 / vAVW03 is also shown in FIG. 16 for the oral swab (16C) and the total excretory swab (16D). The vAVW03 vaccine immunized chickens shed much less virus after challenge compared to the vAVW01 vaccine immunized chickens and also had a lower number of positive swabs.

Table 6: Real-time reverse transcriptase PCR (RRT-) targeting H5N1 viral shedding of eight test groups, matrix genes in oral swabs and total excretory swabs 2, 4, and 7 days (dpc) after H5N1 HPAI challenge PCR)
* v01 = vAVW01 and v03 = vAVW03

FIG. 17 shows the effect of NDV MDA on vAVW03-induced AIV HI titers after vaccine immunization (D21) and challenge (D35) (H5N1 Clade 2.2 (A / Duck / Hungary / 11804/2006) and H5N9 (A / Chicken / Italy / A22 / 1998) using antigen). In the presence of NDV MDA (NDV) on day 21 (after vaccine immunization and before challenge), the average AIV HI titer after vaccine immunization is higher, with detectable HI titers for both antigens There were more chickens. On day 35 (post-challenge), offspring of hens vaccinated with NDV alone had no increase in AIV HI titers after AIV challenge and 10/10 (as opposed to 9/10 in the SPF group) chickens Was defended. In SPF chickens without MDA, a clear increase in AIV HI titer after challenge suggests that the challenge virus replicates to some extent in these chickens. These results suggest unexpectedly good AIV antibody induction and protection in chickens with NDV MDA.
FIG. 18 shows NDV HI titers after vaccine immunization (D21). On day 21, vAVW01 induced NDV titers were usually higher than those induced by vAVW03. On day 21, there is a difference in NDV HI titers between chicks with NDV MDA (from G1 (H5 + NDV) or G4 (NDV) breeders) and those without (from SPF or G5 (H5N1) breeders) Without suggesting, NDV MDA did not interfere with vAVW01 or vAVW03 induced NDV HI titers.
Overall, the results of this experiment show that a single mucosal administration of a relatively low dose (5 log ₁₀ EID ₅₀ ) of the AVINEW engineered variant vAVW03 to 1-day-old chickens provides an excellent level of protection against HPAI H5N1 challenge It clearly shows that The presence of anti-vector (NDV) MDA had no negative effect on AI protection. In contrast, more surprisingly, protection and AI antibody data suggest that there is better AI protection when NDV MDA is present during vAVW03 vaccine immunization in 1-day-old chickens . These results also indicate that AI protection can be induced by vAVW03 in birds with both maternal NDV and AI antibodies.

Protected ducks against H5N1 HPAI of duck chicks induced by engineered AVINEW mutants naturally infect NDV and are also carriers for avian influenza A virus. Even if infected with a highly pathogenic strain, a duck may not necessarily show clinical symptoms after AI infection, but it will transmit the virus to susceptible chickens. This is why ducks are said to be AI Trojans. The purpose of this experiment was to investigate whether the engineered NDV can be used as a vector vaccine for influenza in ducks.
Example 4.1: Duck experiment 1 with 14-day-old Moscoby duck chicks 1
The purpose of this experiment is derived from two different H5N1 clades (vAVW02 expresses the HA gene of clade 2.1 A / chicken / Indonesia / 7/2003, vAVW03 expresses clade 2.2 A / turkey / Turkey 1/2005) (1) Immunogenicity and (2) H5N1 efficacy induced by the two AVINEW-expressing mutants expressing the synthetic HA gene, the efficacy of the parental Avinew strain and normal Moscoby duck It was to compare.
Example 4.1.1: Duck experiment 1: The immunogenicity of the engineered Avinew mutant in duck chicks
One-day-old Moscoby duck chicks were tested for NDV and AIV serology and, surprisingly, 7/10 duck chicks were found to be seropositive for NDV, with an average HI titer of 3.9 log2. there were. All AI HI tests were negative. Another blood sample taken at 13 days of age from 10 ducks showed a negative ND HI titer and the experiment started when the ducks were 14 days old. Three groups were established, each with 10 vaccinated and 7 with contact control ducks. Contact controls were released on the day of vaccination (D0 and D21) and returned to the group the next day. A fourth group of 5 non-vaccinated controls was also included (see Table 7).

Table 7: Duck experiment 1: Group setting for evaluation of immunogenicity of HA-expressing AVINEW mutant

Blood samples were taken at D0, D21 and D42 for the NDV and AI HI tests and the AI SN test (using MDCK cell acclimation M6 11804 H5N1 HPAI Hungary strain). For NDV-specific real-time PCR (primers M4100 and M4220 and probe M4169) (Wise et al. (2004) J Clin Microbiol 42: 329-348) At D12, contact animals were collected at D7 and D12.
The results showed that no adverse reactions were observed and the three vaccines were safe under these conditions. All samples in group 4 (non-vaccinated control) were negative for PCR and serology. All samples of group 1 to 3 non-vaccinated contact birds were negative for PCR and serology, indicating that this vaccine was not transmitted from vaccine-immunized birds to contact animals. The NDV HI titer is shown in FIG. For D21, the HI titer of G1 (3.6 log2; 100% is greater than 3 log2) is significantly higher than that of G2 (100% is less than 3 log2) and G3 (3/10 is greater than 3 log2). However, the mean NDV HI titer 3 weeks after the second dose was similar in the three vaccination groups (5.1, 5.7 and 6.1 log2 in groups 1, 2 and 3, respectively) (ANOVA; p = 0.682) .
AI H5N1 HI titers are shown in FIG. Detectable HI titers (> 3 log2) are found in D21 after the first vaccination for G2 and G3 ducks (except one duck in G2 that had an HI titer of 4 log2) I couldn't. After the second dose, all ducks had HI titers greater than 3 log2 and an average titer of approximately 4 log2. There was no significant difference in AI HI titers induced by the two AVINEW mutants AVW02 and AVW03 at both time points.
AI H5N1 serum neutralization (SN) titers are shown in FIG. Detectable SN titers (greater than 2 log2) are D21 after the first vaccination for G2 and G3 ducks (excluding 2 G3 ducks that had an SN titer of 2 or 3 log2) Was not accepted. After the second dose, all ducks had a HI titer greater than 3 log2 and an average titer of approximately 6.2 log2. There was no significant difference in AI HI titers induced by the two AVINEW mutants AVW02 and AVW03 at both time points.
NDV PCR: NDV PCR results are shown in Table 8. Only a few ducks in groups 1 and 3 were found to be positive after the first and second vaccinations. All positive samples were from pharyngeal swabs except for one swab of group 3 D28. The two ducks that were positive for D25 after the second dose of AVINEW were the only birds that were positive for both D3 and D7 after the first dose. All samples in group 2 were negative despite induction of anti-ND and anti-AI antibodies.

Table 8: Duck experiment 1: NDV real-time PCR test results for pharynx and total excretory swab
(Number of positive ducks / total number)
* All positive samples were from pharyngeal swabs except for one of the three positive birds at group 28 D28 (whose total excretory swab was positive).

  In summary, this experiment confirmed the safety of AVINEW and showed that insertion of the HA gene into the AVINEW-AI mutant did not induce adverse reactions in duck chicks. AVINEW induced significantly higher NDV HI titers after the first dose than the two tested AVINEW-AI mutants, suggesting that HA gene insertion slightly impairs NDV replication. Two instillation doses of 10 chicken doses of the AVW-AI variant were required to induce a positive AI SN titer with positive NDV and AI HI titers in all ducks. Despite the presence of HA genes from two different clades (clade 2.1 for vAVW02 and clade 2.2 for vAVW03), there are differences in the immunogenicity of the two test AVINEW-AI mutants against clade 2.2 H5N1 antigen There wasn't. Only a few birds vaccinated with AVINEW and vAVW03 expelled the virus primarily into the pharyngeal swab. However, this excretion was insufficient to propagate the vaccine virus to contact ducks that remained negative throughout the entire experiment. Several ducks in this experiment were then challenged with H5N1 Hungarian isolates. The result of the challenge is presented in Example 4.1.2.

Example 4.1.2: Duck Experiment 1: H5N1 Protection Induced by Avinew Mutant Manipulated in Moscoby Duck
H5N1 challenge experiments were performed on several ducks vaccinated with vAVW02 or vAVW03 (see Example 4.1.1). Four of the ducks vaccinated twice with vAVW02 or vAVW03 instillation were challenged at 9 weeks of age. Two ducks from Group 1 (AVINEW) were used as negative controls. The vAVW02 and vAVW03 vaccine immunization groups had an average H5 HI titer of 5.5 log2 (4 for 5 log2, 4 for 6 log2) and an average SN titer of 4.0 log2 (1 for 3 log2, 6 for 4 log2, And one was 5 log2SN titer). Eight vaccine immune ducks and two control ducks were challenged by IM administration of 4.7 log10 EID ₅₀ of HPAI H5N1 A / duck / Hungary / 11804/2006 (M6 11804) strain. Total excretory cavities and pharyngeal swabs were obtained 2, 7 and 10 days after challenge, and the heart, pancreas, brain and spleen were sampled at necropsy and examined by PCR and histopathology.
The results are summarized in Table 9. Two controls died within 48 hours after infection. Oral and swallow swabs were H5N1 positive by PCR along with brain, pancreas, heart and spleen. Histopathology of various organs showed signs of hyperacute H5N1 infection. In 8 vaccine immune ducks, no clinical symptoms were observed during the 10-day observation period. Viral shedding was only detected after 3 days of challenge with pharyngeal swabs in 3 out of 8 vaccine immune ducks (2 in G2 and 1 in G3). One of these positive ducks (one of G3) was also positive for AI PCR of the total excretory swab. All other swabs and organs were negative. The lesion was not found in the organ of the vaccine immune duck.

Table 9: Duck experiment 1: Summary of protection results induced by AVINEW vector vaccine in 14-day-old Moscoby duck chicks

  The results showed that the IM challenge was very intense with two control birds. Full clinical protection and partial viral shedding protection were observed in 8 ducks vaccinated in two drops with the AVINEW vector expressing the synthetic H5 gene from the H5N1 isolate.

Example 4.2: Duck Experiment 2 with 1-day-old Moscoby duck chicks 2
The purpose of the duck experiment in this example was to treat one-day-old mosquito duck chicks by one or two administrations of the vAVW03 AVINEW mutant expressing a synthetic HA gene derived from clade 2.2 A / turkey / Turkey / 1/2005. Was to compare the immunogenicity (Example 4.2.1) and efficacy (Example 4.2.2) induced by
Example 4.2.1: Operation in 1-day-old duck chicks Immunogenicity of AVINEW The animals used in this experiment were 1-day-old Moscoby duck chicks, both of which were NDV (HI) and AIV (HI and SN) was found to be negative for serology. Three groups were established, in each of which 10 were vaccine immune ducks and 5 were contact control ducks. Contact ducks were released on the day of vaccination (D0 and D21) and returned to the group the next day. A third group of non-vaccinated controls was also included (see Table 10). Ten vaccinated ducklings from groups 1 and 2 were vaccinated with 50 μL eye drops containing 6.5 log10 EID ₅₀ vAVW03 in D0 (groups 1 and 2) and D14 (group 2 only).

Table 10: Duck experiment 2: Group setting to evaluate the immunogenicity of one or two doses of AVINEW mutant vAVW03 expressing HA

Blood samples are taken at D0, D14 and D35 and HI test is used for NDV (LaSota antigen) and AI (M6 11804 HPAI H5N1 Hungary / 2006 antigen) antibodies and SN test (MDCK cell acclimation M6 11804 H5N1 HPAI Hungary) Used strain).
No adverse reactions were observed, confirming the safety of vAVW03 against 1-day-old Moscoby duck chicks. NDV HI and AI SN titers are presented in FIGS. 22A and B, respectively. D14 shows NDV HI titer that can detect only 10/20 vaccine immune duck 2 weeks after the first administration of vAVW03 (over 3 log2), and also has H5N1 SN titer that only 8/20 can detect Indicated. In D35, HI and SN titers remain the same in Group 1 (6/10 positive in the NDV HI test (mean 2.9 log2), 8/10 positive in the SN H5N1 test (average 1.3) log2)), increased in group 2 after the second dose of vAVW03. All samples from the control group (Group 3) were negative in both the NDV HI test and the H5N1 SN test. All sera from non-vaccinated contact ducks in groups 1 and 2 were also negative for NDV HI and H5N1 SN, except in group 2 where sera were converted for NDV (3 log2) and SN H5N1 (1 log2) Except for one duck. This suggests that the vAVW03 vaccine spread from vaccinated ducks to one of five unvaccinated contact ducks. Following this experiment, several ducks were challenged with H5N1 Hungarian isolates. The result of the challenge is presented in Example 4.2.2.
These results indicated that the safety of vAVW03 in 1-day-old duck chicks was confirmed. A single instillation of 6.5 log ₁₀ EID ₅₀ vAVW03 to 1 day old Moscoby duck chicks only induced low NDV HI and H5N1 SN titers in 40-50% of birds. A clear booster effect on NDV and SN H5N1 titers was observed after the second instillation of vAVW03. In contrast to previous experiments, low anti-NDV and anti-H5N1 antibodies were detected in one non-vaccinated duck in contact with group 2 ducks vaccinated twice with D0 and D14. This suggests that horizontal propagation can occur less frequently under these test conditions.

Example 4.2.2: Ducks Experiment 2: Manipulated AVINEW-AI mutants in 1-day-old duck chicks AI H5N1 efficacy vAVW03 AVINEW-AI mutant expressing the HA gene of clade 2.2 (see Example 4.2.1) H5N1 challenge experiments were performed on several ducks immunized once or twice.
2 non-vaccinated ducks in contact with group 1 and 2 birds and 4 duck from group 1 (1 dose at D0) and 2 (2 doses at D0 and D14) and group 3 Two unvaccinated ducks were challenged at 5 weeks of age. Ten vaccinations, four contacts, and two control ducks were challenged by nasal administration of 4.7 log10 EID ₅₀ of HPAI H5N1 A / duck / Hungary / 11804/2006 (M6 11804) strain. Total excretory swabs and pharyngeal swabs were collected on days 4, 7, and at death. At 10 days after challenge or at death, heart, pancreas, brain, liver and spleen samples were collected at necropsy and examined by PCR and histopathology.
Individual results are shown in Table 11. Two controls from group 2 died within 48 hours after infection. Oral and swab swabs were positive for H5N1 by PCR along with brain, pancreas, heart and spleen. Histopathology of various organs showed signs of hyperacute H5N1 infection. No clinical symptoms were observed in the 10 vaccine immune ducks in groups 1 and 2 during the 10 day observation period. One duck in group 1 (# 402) had no detectable NDV and H5N1 antibodies, and another duck (# 403) only had a low NDV HI titer. This result indicated that these antibody tests could not be fully predictable in terms of clinical protection. Viral shedding was detected in D4 in 3 out of 5 ducks in group 1 (2 in both swabs and 1 in throat swab) and in 1 duck in group 2 only (pharyngeal swab only) . For D7, only one duck from Group 1 and Group 2 was positive for viral shedding in the throat swab. All other swabs and organs were negative. The lesion was not found in the organ of the vaccinated duck. All non-vaccinated ducks maintained in contact with group 1 and 2 vaccinated birds died within 2 or 3 days and, like group 3 control ducks, were positive in PCR analysis on their swabs and organs. (Except one contact duck (# 429) in group 2 that was fully protected). Interestingly, it was this single duck that showed low levels of NDV and H5N1 antibodies when challenged, suggesting that this vaccine was transmitted from the vaccine immune duck to this contact duck. The duck showed no clinical symptoms and its swabs and organs were negative by PCR. An investigation of the histopathological lesions of various organs of the duck that died after challenge included the following: brain (early lymphocytic encephalitis), liver (acute serous showing complex focal necrosis of liver parenchyma) Hepatitis), heart (interstitial edema, myocardial puncture), pancreas (hyperemia, interstitial edema), small intestine (hyperemia, mucosal edema), spleen (hyperemia, lymphocyte depletion in malpiggy bodies), lung ( Hyperemia, interstitial edema, early focal interstitial pneumonia), trachea (mucosal edema). These results indicated mild histopathological changes, including encephalitis, edema in various organs, hyperemia and hepatocyte necrosis.

Table 11: Duck experiment 2: Individual protection results of duck experiment 2 performed with 1 (D0) or 2 (D0 and D14) vAVW03 treatments on 1-day-old Moscoby duck chicks
* T = positive pharyngeal swab; C = positive total excretory swab

  The results show that despite the use of relatively low doses, the oral and nasal challenge is very intense, and this Hungarian H5N1 isolate (isolated from a duck) Suggesting a high level of disease toxicity. Full clinical protection was observed in all vaccinated ducks, which received only one vAVW03 administration at 1 day of age 5 weeks prior to challenge, low or undetectable NDV HI or H5N1 SN The same was true for the ducks that showed only the titer. Viral shedding was observed with fewer ducks (1/5) in group 2 who received 2 vaccinations compared to duck from group 1 (3/5) who received only 1 dose . Interestingly, perhaps only one contact duck that showed detectable NDV and H5N1 antibody titers prior to challenge was fully protected, probably due to horizontal transmission of vAVW03 vaccine from the vaccinated duck.

Example 4.3: Duck Experiment 3 with 1 Day-old Moscoby Duck Chicks 3
The purpose of the duck experiment in this example was to confirm H5N1 protection in 1-day-old SPF Moscoby duck chicks induced by vAVW03 alone or in combination with other vaccines against another H5N1 HPAI isolate. there were.
The experimental design is shown in Table 12. The immunogenicity of a single vAVW03 administration was compared with the immunogenicity of two vAVW03 administrations and the heterogeneous prime-boost immunization schedule. The heterologous prime-boost immunization schedule is 10% of the TROVAC-AIV H5 vector vaccine (fowlpox recombinant vFP89 expressing the unique HA gene of HPAI H5N8 A / turkey / Ireland / 1378/1983 isolate, approved by USA) It consisted of priming with D0 using a chicken dose (approximately 4.5 log10 TCID ₅₀ / dose) by the subcutaneous route followed by administration by the mucosal route of vAVW03 at D14 (see Table 12 for details). D28 and D42 were challenged with 5 ducks from each group with the French HPAI H5N1 Clade 2.2 A / Swan / France / 06299/06 isolate (10 ⁶ EID ₅₀ / duck).

Table 12: Design and defense results of duck experiment 3
* vAVW03 was administered by the intranasal route using 50 μL of vaccine suspension (using mineral water as diluent); TROVAC-AIV H5 was 0.2 mL of vaccine suspension (using Marek vaccine diluent as diluent) Was administered by the subcutaneous route.
** The dose of vAVW03 is log10 EID ₅₀ and the dose of TROVAC-AIV H5 is expressed as log10 TCID ₅₀ ; the dose of TROVAC-AIV H5 corresponds to the 10 chicken dose of a commercial batch of this vaccine.
*** The challenge strain was HPAI H5N1 Clade 2.2 A / Swan / France / 06299/06 / isolate; 6 log10 EID ₅₀ was administered by ocular nasal route; MTD is the mean to death in days It's time.

All non-vaccinated ducks showed clinical symptoms and died within 4 days after challenge. None of the vaccinated ducks showed clinical symptoms or died (see Table 12). Oropharyngeal swabs and total excretory swabs were collected at various times after challenge (2.5, 4.5, 6.5, 9.5 and 11.5 days after challenge). Based on the paper by Spackman et al. (J Clin Microbiol, 2002, 40: 3256-3260), viral load was measured by real-time RT-PCR based on M. The results of the D28 and D42 challenge are presented in FIGS. 23a-d and 24a-d, respectively.
Viral shedding data show that vaccinated ducks shed less virus than unvaccinated controls, and the percentage of positive birds after D28 (Figure 23a-d) and D42 (Figure 24a-d) challenge also decreased. Clearly shown. There was no difference between the samples in groups 2 and 3, indicating that one vAVW03 administration at 1 day of age under these conditions provided the same protection as two vAVW03 immunizations at D0 and D14 . The positive rate, as well as the viral load, was lower compared to those in groups 2 and 3, especially in the D28 challenge with group 4 ducks on the heterogeneous prime-boost immunization schedule. These results show that priming with a fowlpox recombinant that expresses another HA gene from the same subtype administered prior to vAVW03 provides a level of protection compared to one or two doses of vAVW03. It shows improvement.
AI HI titers are serum samples taken before (D28 and D40) and after (D42 and D57) challenge with D28 and D42, respectively, and HPAI H5N1 (French strain 06167i H5N1 closely related to the challenge strain) as an antigen. Measured using Clade 2.2.1) (see Table 13). Only a few ducks in group 4 had detectable HI titers with D28. It would be meaningful to state that all vaccinated ducks were protected from intense HPAI challenges despite no detectable serum conversion to AI upon challenge. This result confirmed previous results that showed that the HI test cannot be used to predict the effectiveness of such engineered NDV AI vaccines.
Fourteen days after D28 challenge, HPAI H5N1 HI titers increased from 0 in D28 to 7.2 log2 in D42 in Groups 2 and 3, but increased from 2.3 (D28) to 6.0 (D42) in Group 4. Compared to groups 2 and 3, the lower post-challenge HI titer in group 4 suggests that the challenge virus replicated little in this group. Such a decrease in challenge virus replication in group 4 ducks was observed in virus shedding data after challenge with D28 (see above and FIGS. 23a-d).

Table 13: Duck Experiment 3: Average AI HI titer before and after challenge
In summary, single (D0) or twice (D0 and D14) mucosal administration of vAVW03 to 1-day-old Moscoby duck chicks protects them against the HPAI H5N1 challenge performed at D28 and D42. did. Heterologous priming with fowlpox recombinants (expressing HA from H5N8 isolate) prior to vAVW03 administration improved protection against early challenge at D28.

Example 4.4: Duck experiment 4: Duration of immunity in 1-day-old Moscoby duck chicks The purpose of the duck experiment in this example was the TROVAC-AIV H5 / vAVW03 xenotype tested with a single dose of vAVW03 or in the duck experiment 3 It was to determine the duration of immunization induced by the prime-boost immunization schedule.

Table 14: Duck experiment 4 design and clinical defense results
¹ : Number of ducks challenged with D65 + D86 (MTD: average time to death in days)
² : vAVW03 was administered by the intranasal route using 50 μL of vaccine suspension (using mineral water as diluent); TROVAC-AIV H5 was administered by subcutaneous route using 0.2 mL of vaccine suspension; inactivated The vaccine is a commercial oil adjuvanted inactivated vaccine. Re5 reverse genetics H5N1 isolate containing HA (modified at the cleavage site) and NA gene from Clade 2.3 A / duck / Anhui / 1/2006 isolate Including.
³ : The dose of vAVW03 is log10 EID ₅₀ and the dose of TROVAC-AIV H5 is expressed as log10 TCID ₅₀ ; the dose of TROVAC-AIV H5 corresponds to the 10 chicken dose of a commercial batch of this vaccine.
⁴ : Number of birds immunized from morbidity-Number of birds evaded death / total (MTD: average time to death in days)
⁵ : Challenge strain was HPAI H5N1 Clade 2.2 A / Swan / France / 06299/06 / isolate; 6 log10 EID ₅₀ was administered by ocular nasal route.

None of the ducks produced detectable AI HI antibodies against HP H5N1 06167i clade 2.2.1 antigen (related to challenge strain) prior to challenge.
These late challenges were effective. This is because the challenge induced 100% morbidity and killed most of the non-vaccinated control ducks (one morbid duck survived the late D86 challenge; see Table 14). Most birds vaccinated with vAVW03 were protected. That is, only 1/8 had mild clinical symptoms, survived D65 challenge, and 1/9 died of clinical symptoms in D86 challenge. All birds in the Prime-Boost (DRO TROVAC AIV-H5 and D15 vAVW03) groups survived both challenges, and only one bird showed clinical symptoms in an earlier (D65) challenge. None of the Group 4 birds immunized with the inactivated Re5 vaccine showed clinical signs and died.
Oropharyngeal and total excretory virus excretion were also examined after challenge. All control ducks expelled the virus at high levels (EID ₅₀ comparable to 6.8 and 4.9 for the oropharynx and total excretion route, respectively) for at least 9.5 days (for surviving birds). In groups 2 and 3, the emission levels were lower (about 1 and 2 log10 lower respectively) and decreased more rapidly than the control group. Group 4 emission profiles were close to those observed in Group 3.
In conclusion, a single dose of vAVW03 on Moscowby duck chicks (2 days old) provided a good level of protective immunity up to 84 days of age. A prime-boost immunization schedule with fowlpox vector followed by NDV vector provides a better protective immune response than a single dose of NDV vector and a protective response similar to inactivated Re5 vaccine.

Example 4.5: Duck Experiment 5: Efficacy of vAVW03 in Peking Duck Chicks The purpose of this experiment was to determine the efficacy of vAVW03 against Peking Duck HPAI H5N1 challenge. Seven-day-old Peking Duck chicks were vaccinated as shown in Table 15. The challenge strain was an HPAI H5N1 clade 2.2 A / turkey / Turkey 1/2005 isolate. Administration of 6 and 7 log10 EID ₅₀ by ocular nasal route was used for D28 and D42, respectively. Clinical symptoms (morbidity) and death (mortality) were recorded after challenge. Viral shedding after challenge was measured by real-time RT-PCR of buccal and total excretory swabs taken on days 2, 5 and 8 after challenge (dpc). Experimental design and protection results are shown in Table 15.

Table 15: Design and defense results of duck experiment 5

¹ : vAVW03 was administered by 50-L vaccine suspension (using mineral water as diluent) by ocular nasal route; TROVAC-AIV H5 was 0.2 mL vaccine suspension (using Marek vaccine diluent as diluent) ) Was administered by the subcutaneous route.
² : The dose of vAVW03 is log10 EID ₅₀ and the dose of TROVAC-AIV H5 is represented as log10 TCID ₅₀ ; the dose of TROVAC-AIV H5 corresponds to the 10 chicken dose of a commercial batch of this vaccine.
³ vFP89: Chickenpox vector AIV H5 (see US 2008/0107681 and US 2008/0107687)
⁴ Re5: Oil adjuvant added inactivated vaccine based on reverse genetics strains containing modified HA gene and NA gene derived from HPAI H5N1 A / duck / Anhui / 1/2006 (Clade 2.3).
⁵ : Number of chickens positive in buccal or total excretory swabs at 2, 5 or 8dpc.

  These results indicated that Peking Duck was relatively resistant to H5N1 challenge. This is because only about half of the unvaccinated birds showed clinical symptoms or died after challenge. Nevertheless, most of them expelled detectable amounts of virus and showed active replication of challenge strains. All vaccinated ducks were clinically protected on both challenge days, and the number of birds shedding virus was reduced compared to non-vaccinated controls. These results show that vAVW03 can induce significant defense in Peking Duck.

Example 4.6: Duck Experiment 6: Efficacy of vAVW03 in 1-Day-old Peking Duck Chicks with NDV MDA The purpose of this duck experiment 6 was to assess the effects of NDV MDA on vAVW03-induced efficacy. It was to evaluate the HPAI H5N1 efficacy induced by a single dose of vAVW03 in Peking duck born by breeding birds vaccinated with an inactivated combination vaccine containing. A 2 day old Peking duck with NDV MDA was used in this experiment. SPF moscoby duck chicks were also used to demonstrate the effectiveness of the challenge. Experimental design and protection data are presented in Table 16.

Table 16: Design of duck experiment 6 and results of protection against HPAI H5N1 challenge
* ON = eye nose
** Challenge strain was HPAI H5N1 Clade 2.2 A / Swan / France / 06299/06 isolate; 6 log10 EID ₅₀ was administered to D24 by ocular nasal route; MTC averaged to clinical symptoms expressed in days MTD is the average time to death in days.
The HPAI H5N1 challenge was evidenced by the rapid mortality of unvaccinated Moscoby duck chicks used as controls (all died 3.5 days after challenge). However, the unvaccinated Peking Duck was much more resistant to the H5N1 challenge. Because only 3/5 showed clinical symptoms, only 1/5 died 6.5 days after challenge. Partial protection against morbidity (67-89%) was induced by vAVW03, with no clear dose or route effects in Peking Duck.
All control Peking Duck drained the virus through the oropharynx. Virus release was reduced for virus load, release time and number of positive birds in the vaccinated group.
This experiment demonstrates that a single dose of vAVW03 can induce AI protection in Peking Duck with NDV MDA.

Although preferred embodiments of the present invention have been described in detail as described above, the present invention as defined above is described above since many obvious variations thereof are possible without departing from the scope of the present invention. It will be understood that the invention is not limited to the specific matter.
All documents cited or referenced in this application's cited documents, and all documents cited or referenced in this document ("Documents cited in this document"), as well as all documents cited or referenced in this document's cited documents The document is hereby incorporated by reference together with any manufacturer's instructions, descriptions, product specifications, and product instructions for any product described in this document or any document included by reference in this document. And can be used in the practice of the present invention.

Claims (25)

  A composition comprising an engineered NDV vector.   2. The composition of claim 1, wherein the engineered NDV vector comprises a heterologous polynucleotide encoding an avian influenza HA antigen or variant thereof.   The engineered NDV vector has at least 90% sequence identity with the polynucleotide having the sequence shown in SEQ ID NO: 1 or at least 90% sequence identity with the polynucleotide having the sequence shown in SEQ ID NO: 1 The composition of Claim 1 or 2 containing the polynucleotide complementary to the polynucleotide which has sex.   4. The composition of claim 2 or 3, wherein the avian influenza HA antigen is an H5 HA polypeptide.   The composition according to any one of claims 2 to 4, wherein the HA antigen has a modified cleavage site.   6. The composition of any one of claims 2-5, wherein the HA antigen has at least 80% amino acid sequence identity with a polypeptide having the sequence shown in SEQ ID NO: 15, 17, 19, 21, or 23.   7. The composition of any one of claims 2-6, wherein the heterologous polynucleotide has at least 80% sequence identity with a polynucleotide having the sequence shown in SEQ ID NO: 14, 16, 18, 20, or 22.   A method for producing an engineered NDV vector by modifying the genome of NDV, comprising the step of introducing a polynucleotide into the non-essential region of the NDV genome.   A polynucleotide in which NDV has at least 90% sequence identity with the polynucleotide having the sequence shown in SEQ ID NO: 1, or a polynucleotide having at least 90% sequence identity with the polynucleotide having the sequence shown in SEQ ID NO: 1 9. The method of claim 8, comprising a polynucleotide complementary to.   10. The method according to claim 8 or 9, wherein the non-essential region is selected from a region consisting of an untranslated region located upstream of the NP gene, between two genes of the APMV genome, and downstream of the L gene.   The method according to any one of claims 8 to 10, wherein the non-essential region is located between the P and M genes or between the M and F genes.   A method of inducing or immunizing a bird against at least one avian pathogen, the method comprising administering an engineered NDV vector expressing at least one antigen.   A polynucleotide in which the NDV vector has at least 90% sequence identity with the polynucleotide having the sequence shown in SEQ ID NO: 1 or at least 90% sequence identity with the polynucleotide having the sequence shown in SEQ ID NO: 1 13. The method of claim 12, comprising a polynucleotide complementary to the nucleotide.   14. The method according to claim 12 or 13, wherein the antigen is an avian influenza antigen.   15. The method of claim 14, wherein the avian influenza antigen is hemagglutinin (HA).   16. The method of claim 15, wherein the avian HA antigen has at least 80% sequence identity with a polypeptide having the sequence shown in SEQ ID NO: 15, 17, 19, 21 or 23.   17. The method according to any one of claims 12-16, wherein administration is by eye drops, spray, drinking water, egg inoculation, intramuscular or subcutaneous administration.   18. A method according to any one of claims 12-17, wherein the administration is a prime-boost.   A vaccine or composition comprising a prime-administration of a composition according to any one of claims 1-7 and a recombinant viral vector comprising an avian pathogen antigen and expressing it in vivo, or An inactivated virus vaccine comprising an avian pathogen antigen, or a vaccine or composition comprising an avian pathogen antigen (subunit), or a boost-administration of a DNA plasmid vaccine or composition comprising and expressing an avian pathogen antigen. The method according to 18.   A vaccine or composition comprising a recombinant viral vector in which prime-boost contains and expresses an avian pathogen antigen in vivo, or an inactivated virus vaccine comprising an avian pathogen antigen, or a vaccine comprising an avian pathogen antigen (subunit) Or a prime-administration of a DNA plasmid vaccine or composition comprising and expressing an avian pathogen antigen and a boost-administration of a composition according to any one of claims 1-7. The method according to 18.   The composition of any one of claims 1-7, wherein the prime-administration comprises a composition comprising a poxvirus comprising and expressing an avian influenza antigen, and the boost-administration comprises an avian influenza antigen. 21. The method of claim 20, comprising.   The prime-boost comprises prime-administration of a composition according to any one of claims 1-7 and boost-administration of a composition according to any one of claims 1-7. The method described.   23. A method according to any one of claims 12-22, wherein the bird is a chicken or duck.   An engineered NDV vector comprising a polynucleotide having at least 90% sequence identity to SEQ ID NO: 1. A polynucleotide selected from the group consisting of:
a) a polynucleotide having at least 90% sequence identity to a polynucleotide having the sequence shown in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12 or 24;
b) a polynucleotide having at least 90% sequence identity with a polynucleotide encoding a polypeptide having the sequence shown in SEQ ID NO: 3, 5, 7, 9, 11 or 13;
c) A polynucleotide complementary to the polynucleotide described in a) or b).